Exam 3 Flashcards

1
Q

Where in a cell does splicing occur?

A

The nucleus

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2
Q

Where in a cell does transcription occur? (eukaryotes)

A

The nucleus

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3
Q

Where in a cell does translation occur? (eukaryotes)

A

The cytoplasm

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4
Q

Definition: Translation

A

Translating the ‘language’ of nucleic acids into the language of proteins (nucleotides into amino acids)

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5
Q

What is codon usage bias?

A

Different species prefer different codons for the same amino acid

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6
Q

True or False: The AUG codon for methionine is the most common start codon in both eukaryotes and prokaryotes

A

TRUE

–> In prokaryotes, a specialized form of methionine is used for the initiating methionine known as N-formyl methionine, though

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7
Q

True or False: The start codon in translation signifies the C terminal of the protein

A

FALSE: The start codon signifies the N-terminal of the protein

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8
Q

What are the three stop codons?

A

UAA, UAG, UGA

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9
Q

What is the difference between methionine (Met) and N-formylmethionine (fMet)?

A

N-formylmethionine has a H-C=O group on its N-group

–> Draw this!

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10
Q

True or False: Despite the slight difference between Met and fMet, eukaryotes can’t recognise fMet as foreign

A

FALSE: The eukaryotic immune system recognizes proteins with fMet as foreign

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11
Q

Where is fMet used (which types of cells)?

A

In prokaryotes, mitochondria, and chloroplasts

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12
Q

What establishes the reading frame in translation?

A

The initiator AUG

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13
Q

Definition: Reading frame

A

The reading frame is the phase in which 3 nucleotides are read into amino acids. There are 3 potential reading frames and the AUG specifies which frame is used.

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14
Q

Definition: Open reading frame

A

An open reading frame is the series of codons that come between the initiation AUG and the first Stop codon

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15
Q

What are the three types of point mutations?

A

Silent, Missense, and Nonsense

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16
Q

Describe a silent mutation

A

A point mutation that does not end up making a difference in the amino acid that is coded (due to redundancy of the genetic code)

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17
Q

Describe a missense mutation

A

A point mutation that results in one amino acid being changed

–> Normally not that big of a problem

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18
Q

Describe a nonsense mutation

A

A point mutation that changes a regular codon to a stop codon, resulting in premature ending of the protein

–> Usually results in a loss of function, may or may not be a big deal

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19
Q

What are tRNAs?

A
  • A class of noncoding RNAs made by RNA Pol III

- Adapters that link the sequence of the RNA to the sequence of the protein

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20
Q

What is at the 3’ end of tRNA?

A

A single-stranded ‘CCA’ at the 3’-end that links to the amino acid

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21
Q

What is the anticodon of tRNA?

A

A sequence of 3 nucleotides that can base pair with the codon of the mRNA

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22
Q

How does tRNA fold into its cloverleaf shape?

A
  • By intramolecular base pairing with 4 regions that are double-helical
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23
Q

True or False: tRNAs have about 10% of their bases modified before they leave the nucleus

A

TRUE

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24
Q

Why are tRNAs chemically modified?

A

Modifications are crucial for tRNA structure, function, and stability

  • Modifications of specific positions affect the behavior of tRNAs during translation
  • Some modifications, such as pseudouridines, make the tRNA more rigid whereas others such as dihydrouridines make it more flexible
  • Some modifications are essential for ‘charging’ the tRNA with correct amino acid
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25
Q

True or False: Hypermodified tRNAs are targeted for degradation

A

FALSE: HYPOmodified tRNAs are targeted for degradation

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26
Q

What does Wobble Base Pairing do?

A

Allows some tRNAs to recognize more than 1 codon because it doesn’t require a perfect match in the 3rd position of the codon
–> i.e. non-standard Watson-Crick pairing

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27
Q

What is the first step of decoding the genetic code? What enzyme does this?

A
  • Linking the correct amino acid to the tRNA is the first step
  • This is done by aminoacyl tRNA synthetases
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28
Q

What is an aminoacyl tRNA?

A

A tRNA with the amino acid attached to it

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29
Q

Describe the bond that forms between the amino acid and the 3’ end of tRNA

A
  • An ester bond forms between the acid of the amino acid and the 3’ hydroxyl of the ribose at the 3’-end of the tRNA.
  • This is a high energy bond.
  • This energy is used later to link the amino acid to the growing protein chain.
  • Because of the high energy bond, an amino acid linked to a tRNA is sometimes said to be ‘activated’
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30
Q

What is an adenylated amino acid?

A

An amino acid that is prepped to be added to a tRNA

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31
Q

True or False: One ATP equivalent is needed to esterify a tRNA

A

FALSE: TWO ATP equivalents are needed to esterify a tRNA

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32
Q

Describe the steps taken to attach an amino acid to a tRNA

A
  • The energy of ATP hydrolysis (ATP –> AMP, so 2 phosphates taken off) is used to attach the AA to AMP, forming an adenylated amino acid.
  • An ester linkage forms between the adenylated AA and the 3’-OH of the tRNA. This yields the activated AA
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33
Q

How do aminoacyl tRNA synthetases recognize their cognate tRNAs?

A

Aminoacyl tRNA synthetases recognize their cognate tRNAs by interacting primarily with the anticodon and the acceptor stem

Aminoacyl tRNA synthetases have 2 separate pockets that must both recognize the amino acid

  • -> Active site pocket that attaches the AA (preferential binding to correct AA)
  • -> Editing (proofing) pocket that exclude the correct AA, while allowing access by closely related AA, if incorrect AA binds while adenylated or bound to tRNA, the bond is hydrolysed
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34
Q

Describe the functions of the proofreading pocket of aminoacyl tRNA

A
  • The correct AA has the highest affinity for the active site pocket of its specific tRNA. The pocket also eliminates most AA that are too large. However, it is not as good at eliminating those that are too small or are closely related (ex: Leu and Ile)
  • After the amino acid is esterified to its tRNA, it is switched to a second pocket for editing/proofreading. This involves hydrolysis of the incorrect bond. Editing (proofing) pocket excludes the correct AA, while allowing access by closely related AA, if incorrect AA binds while adenylated or bound to tRNA, the bond is hydrolysed
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35
Q

What are the two enzymatic activities of tRNA synthetases?

A

i) Synthesis of the amino acid to the tRNA

ii) A proofreading activity

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36
Q

How does the protein grow after tRNA with the proper amino acid attached has bound to mRNA?

A
  • The peptide grows from the NH2 to the COOH terminus
  • The peptide bond is formed by the nucleophilic attack of the amino group of the incoming aminoacyl-tRNA on the ester bond of the peptidyl tRNA (tRNA with a growing peptide chain attached to it)
  • Energy is provided by the high energy ester bond in the aminoacyl tRNA.
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37
Q

What is the bacterial ribosome made up of? (#S rRNA)

A

-70S rRNA made up of 50S and 30S subunits

–> 16S rRNA in the 30S subunit can be checked for presence of bacteria

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38
Q

What is a eukaryotic ribosome made up of? (#S rRNA)

A

-80S rRNA made up of 60S and 40S subunits

–> 18S rRNA in the 40S subunit can be checked for presence of eukaryotic cells

–> Also 28S rRNA comes from the 60S subunit

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39
Q

What are the names of the binding pockets in a ribosome?

A

A- Aminoacyl-tRNA
P- Peptidyl-tRNA
E- Exit site

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40
Q

What is a ribozyme?

A

A mix of RNA and protein

–> ribosomes are ribosymes, with 2/3 RNA and 1/3 protein

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41
Q

What do rRNAs do?

A
  • Responsible for ribosome’s overall structure
  • Ability to position tRNAs on the mRNA
  • Ribosome’s catalytic activity in forming peptide bonds
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42
Q

True or False: Ribosomal proteins bind to the outside of the rRNA mass with protrusions into the rRNA mass and are not direct participants in catalysi

A

TRUE

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43
Q

Describe the five steps of translation initiation in prokaryotes

A

1) Small ribosomal subunit binds to the mRNA
2) A special initiator tRNAmet (met-tRNAiMet), a tRNA with Met or Formyl-Met in bacteria covalently bound to it, is brought into the P site
- – AUG is the only codon in for which there are two tRNAs, an initiator Met-tRNAimet and a Met-tRNAmet for internal Met codons.
3) The initiating start codon is recognized
4) The large ribosomal subunit joins the small subunit
5) The tRNA with the 2nd amino acid is brought in to the A site

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44
Q

Describe start codon recognition in prokaryotes

A
  • Requires Shine-Dalgarno sequence
  • -> This is because bacteria have no 5’ cap to to direct ribosome binding
  • The 3’ end of the 16S rRNA base pairs with the Shine-Dalgarno sequence
  • -> The nearest AUG downstream of this binding site is the beginning of translation
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45
Q

True or False: The Shine-Dalgarno sequence is very well conserved

A

FALSE: It’s really poorly conserved!

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46
Q

What accounts for the polycistronic nature of bacterial mRNAs?

A

There are multiple Shine-Dalgarno sequences in a bacterial mRNA, meaning there are multiple ribosome binding sites and therefore many different proteins can be made from only one mRNA

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47
Q

What is the advantage of having polycistronic mRNAs in prokaryotes?

A

Having all components of a metabolic pathway can be regulated together (i.e. lac operon)

–> Also, smaller genome

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48
Q

True or False: The start codon in eukaryotic mRNAs is a part of the consensus sequence (part of the Kozak sequence)

A

TRUE! The start codon is NOT downstream of the consensus sequence like it is in prokaryotes

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49
Q

True or False: Most eukaryotic mRNAs are polycistronic

A

FALSE: Most eukaryotic mRNAs are NOT polycistronic (most prokaryotic mRNAs ARE polycistronic though)

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50
Q

Describe eukaryotic translation initiation (basic)

A

-In eukaryotes, the small ribosomal subunit with met-tRNAimet starts at the 5’-Cap and scans the mRNA for a start codon located within an optimal setting, i.e. with a good Kozak sequence. Thus, 5’-UTRs can vary greatly in length and the first AUG is not necessarily the start site for translation, although it is for ~90% of the mRNAs

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51
Q

Describe eukaryotic translation (in-depth)

A
  • Initially, Met-tRNAimet is loaded onto the small subunit along with initiation factors such as eIF2, which binds GTP to use for energy. eIF2 delivers Met-RNAimet to the P site.
  • Of all the tRNAs, only Met-tRNAimet is capable of binding to the small ribosome without the complete ribosome.
  • This complex then binds to the 5’end of the mRNA, which is recognized because of the eIF4E & eIF4G proteins bound to the 5’-Cap.
  • Notes:
  • – The little “e” stands for eukaryotic. Similar initiation factors exist in prokaryotes.
  • – The mRNA must have both a Cap and a poly(A) tail to be recognized for translation.
  • – There are many more initiation factors.
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52
Q

Describe eukaryotic assembly of the full ribosome

A
  • After the small subunit binds to the mRNA, it scans for the first AUG in a good context, i.e. the Kozak sequence
  • eIF2 hydrolyzes GTP when contact is made with the AUG start codon.
  • After locating the initiating AUG, eIF2 and other initiation factors dissociate, and the large subunit binds.
  • The first aminoacyl-tRNA binds as specified by the codon and the first peptide bond is formed.
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53
Q

What is the first elongation factor to bind in prokaryotic translation?

A

EF-Tu

EF-Tu (elongation factor thermo unstable) is a prokaryotic elongation factor responsible for catalyzing the binding of an aminoacyl-tRNA (aa-tRNA) to the ribosome. It is a G-protein, and facilitates the selection and binding of an aa-tRNA to the A-site of the ribosome. As a reflection of its crucial role in translation, EF-Tu is one of the most abundant and highly conserved proteins in prokaryotes.

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54
Q

Describe eukaryotic/prokaryotic elongation (in-depth)

A
  1. EF-Tu in bacteria and EF1 in eukaryotes deliver aminoacyl tRNAs to the A site.
  2. The accuracy of the anticodon-codon interaction is verified by proofreading
  3. When accurate, a new peptide bond is catalyzed by peptidyl transferase (= 28S rRNAin 60S large subunit of eukaryotes, 23S rRNAin 50S large subunit in prokaryotes)
  4. Large subunit translocation: The large subunit moves and shifts the tRNAs into the E- and P-sites. This is facilitated by EF-G in bacteria and EF-2 in eukaryotes
  5. Small subunit translocation: The small subunit plus its bound mRNA shift 3 nucleotides over and the A site is available again. This is called indexing.
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55
Q

What does EF-2 do in translation? What does EF-Tu do?

A

EF2: Involved in indexing

EF-Tu: Involved in bringing in the aminoacyl tRNA

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56
Q

How many ATP equivalents are needed for each peptide bond formation in translation?

A

Four!

  • 2 high energy phosphates for the aminoacyl tRNA synthetase to adenylate the AA
  • 1 high energy phosphate - EF-Tu/EF1~GTP -
  • 1 high energy phosphate - EF-G/EF2~GTP -
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57
Q

Which of the given statements regarding the initiation of translation in eukaryotes is/are true:

a. The first AUG in the mRNA is the initiator AUG.
b. The large ribosomal subunit is responsible for identifying the initiating AUG
c. The small and large ribosome subunits bind simultaneously to the mRNA to begin translation.
d. The mRNA must have eIF2, eIF4E, eIF4G, and polyA binding proteins bound to it before initiation occurs.

A

D) The mRNA must have eIF2, eIF4E, eIF4G, and polyA binding proteins bound to it before initiation occurs.

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58
Q

What does protein chain termination require?

A

-Protein release factors (these are proteins themselves)

–> These bind where there is a stop codon, because no tRNAs recognize stop codons

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59
Q

How does a protein release factor terminate translation?

A
  • The release factors cause hydrolysis of the last peptide bond.
  • Hydrolysis of that bond causes dissociation of the ribosomes from the mRNA
  • The ribosomes are then free to initiate translation of another mRNA Peptide
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60
Q

True or False: Protein release factors are structurally distinct from tRNAs

A

FALSE: Protein release factors perform molecular mimicry, and look very similar to tRNAs in structure (also makes sense b/c they need to look like tRNAs in order to get into the ribosome)

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61
Q

What is a polysome/polyribosome?

A

An mRNA covered with multiple ribosomes is called a polysome or polyribosome

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62
Q

True or False: A Single mRNA May Be Translated Simultaneously by Many Ribosomes

A

TRUE

–> Called a polysome

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63
Q

True or False: Only prokaryotes use polysomes

A

FALSE: Both prokaryotes AND eukaryotes use polysomes

–> This dramatically increases the amount of protein made from a single mRNA

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64
Q

What is ribosome recycling?

A

When a protein is done being made, the ribosome dissociates, but because of the close proximity of the end of the protein and the beginning of the translation site, the ribosome goes right back and attaches at the beginning to synthesize another protein

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65
Q

What is read-through (translation)?

A

When an amino acid is inserted where a stop codon is

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66
Q

What is programmed translational recoding?

A

Deliberate alteration of the reading of the mRNA caused by the structure of the mRNA (stem loops, pseudoknots)

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67
Q

Describe programmed translational recoding

A
  • Recoding alters the reading of individual codons and thus alters the colinearity between the mRNA and the protein
  • Recoding occurs due to structural elements in the mRNA
  • Recoding can occur in up to 80% of the translation of a particular mRNA
  • Can have both -1 and +1 frameshifting (or +2) of the ribosome depending on the mRNA. Can get up to 4 different proteins from 1 mRNA!
  • -> Often called programmed frameshifting or programmed ribosomal frameshifting.
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68
Q

What amino acid can be created from serine via programmed read-through?

A

Selenocystiene

–> This insertion requires a specific selenocysteine translation factor

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69
Q

Why is translational frameshifting so important to viruses?

A
  • In HIV and some other viruses, a stop codon is bypassed by a translational frameshift, enabling the virus to make the reverse transcriptase (polymerase) needed for the virus to replicate its genetic content. Without translational frameshifting there would be no HIV AIDS
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70
Q

Would you classify the gag/polymerase mRNA in HIV as being polycistronic or monocistronic? Why?

A

Tends to be called polycistronic because there are 2 proteins made. However, there is only one RNA, so it could be argued that it is monocistronic

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71
Q

How does tetracycline work?

A

Blocks binding to the A site in translation

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72
Q

How does streptomycin work?

A

Prevents transition from the initiation to elongation stage

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73
Q

How does chloramphenocol work?

A

Blocks peptide bond formation

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74
Q

How does erythromycin work?

A

Blocks translocation (indexing)

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75
Q

How does puromycin work?

A
  • It Mimics an Amino Acyl tRNA
  • Puromycin is a mechanism based inhibitor. It enters the A site, forms a peptide bond, but cannot elongate further because it has an amide where it should have an ester
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76
Q

True or False: In eukaryotes, only the 5’ cap (not the poly-A tail) is needed to initiate translation

A

FALSE: Both the 5’-Cap and the poly-A tail are required to initiate effective translation

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77
Q

What is nonsense-mediated mRNA decay?

A

Nonsense-mediated mRNA decay degrades mRNAs with a nonsense (stop) codon in the wrong place, which usually occurs because of improper splicing

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78
Q

What post-transcriptional modifications must mRNA have in order to be transported to the nucleus (eukaryotes obviously)?

A
  • CBC: Cap binding complex
  • EJCs: exon junction complexes
  • PABPs: poly-A binding proteins
  • Nuclear Export Receptor
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79
Q

What must be removed from mRNA before it can be transported to the cytoplasm (eukaryotes)?

A

Nonsense codon in frame upstream of an exon junction complex

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80
Q

Describe the Steps In Nonsense-Mediate mRNA Deca

A
  • When the 5’-end of the mRNA emerges from the nuclear pore, a ribosome begins a ‘test’ round of translation.
    • As the ribosome moves along the mRNA, it displaces the exon junction complexes (EJCs) that are bound to each splice site.
    • The normal Stop codon will be in the last exon and there will be no more EJCs, so the mRNA passes inspection and is released to the cytosol for typical translation.
    • However, if the ribosome reaches a Stop codon while there are still EJCs, that mRNA is rapidly degraded.
    • Thus, the first round of translation checks the ‘fitness’ of the mRNA for translation
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81
Q

When is nonsense-mediated decay triggered?

A
  • Nonsense-mediated decay is triggered when a Stop codon is found before all the EJCs are stripped off.
  • -> This is elicited by the binding of Upf proteins to each remaining EJC. However, this mechanism is triggered only when the aberrant Stop codon is in the same reading frame as that of the normal protein

–> Is 5’ exonucleolytic decay (starts at 5’ end)

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82
Q

True or False: Translation requires coordination between the mRNA, tRNAs, and the ribosome.

A

TRUE

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83
Q

What do newly synthesized proteins require before they are functional?

A
  • Correct folding (conformation)
  • Cellular localization (protein trafficking)
  • Proteolytic processing
  • Modifications
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84
Q

What is Co-Translational Protein Folding?

A

Proteins Folding into Subdomains as They Are Extruded from the Ribosome

–> This folding is spontaneous as a result of noncovalent thermodynamic forces (hydrogen bonding, electrostatic interactions, van der Waals forces).

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85
Q

How do chaperone proteins work?

A
  • Chaperone proteins reversibly bind to hydrophobic regions of nascent proteins and proteins in intermediate stages of folding.
  • Chaperones can stabilize folding intermediates, promote correct folding, maintain proteins in an unfolded state so they can cross membranes, help unfold misfolded segments, prevent misfolding, and prevent protein aggregation.
  • -> A big function of chaperones is to unfold proteins that are destind for degradation.
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86
Q

Describe ‘molten globule’ proteins

A

Each domain of a nascent protein quickly attains a molten globule (loosely folded) state as a result of non-covalent interactions.

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87
Q

Compare and contrast Hsp 60 and Hsp 70

A
  • Both hsp60 and hsp70 have a high affinity for hydrophobic patches on polypeptide chains and both utilize ATP to regulate protein folding.
  • However, the hsp60 and 70 proteins function differently. Hsp 70 acts early in the life of many proteins, often before they leave the ribosome, and hsp60 acts after they are released
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88
Q

Describe Hsp70’s mechanism

A
  1. Each hsp 70 binds to stretches of 4 – 5 hydrophobic amino acids as the protein exits the ribosome.
  2. The hydrolysis of ATP helps promote conformational changes
  3. The binding of a new ATP to hsp70, causes hsp 70 to dissociate from the protein
  4. This cycle can repeat a number of times.
  • -> Occurs while the protein is being expelled from the ribosome
  • -> Only binds to hydrophobic regions
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89
Q

What does a chaperonin do?

A
  • Chaperonins sequester proteins to allow them time to refold by forming a “basket” with hydrophobic sides
  • Confinement of a protein in the basket gives the protein time to refold without interference from other cellular proteins. Confinement lasts for ~15 seconds
  • If the protein isn’t correctly folded the first time, the cycle repeats.
  • The cap on the basket requires ATP
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90
Q

What is a proteasome?

A

A protease complex that degrades misfolded proteins or other proteins that are targeted for destruction by ubiquitinatio

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91
Q

Describe the structure of a 26S proteasome

A
  • 19S Regulatory ‘cap’. Acts as a gate. Recognizes the protein to be degraded and threads it through this cap, which unfolds the protein so it can be digested.
  • -> Where the hydrolase is
  • 20S Catalytic core contains four stacks of heptamericrings with protease activity
  • -> Proteases face the inside so they can’t degrade all proteins in the cell!
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92
Q

True or False: Proteasomes are only present in the cytoplasm of the cell

A

FALSE: Proteasomes are present in the cytoplasm AND the nucleus of cells (if it’s a nucleated cell)

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93
Q

True or False: Proteases are processive

A

TRUE: They clip proteins every 6 to 7 amino acids

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94
Q

What is ubiquitin?

A

Ubiquitin is a 76 amino acid protein that is highly conserved among eukaryotes

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95
Q

What is an isopeptide bond?

A

An isopeptide bond is an amide bond that is not present in the main chain of a protein. The bond forms between the carboxyl terminus of one protein (ubiquitin) and the amino group of a lysine residue on another (target) protein

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96
Q

When is a protein recognized by the 19S cap of the proteasome (for degradation)?

A

When the linkage is between Lys48 of one ubiquitin and Gly76 of another ubiquitin and when there are at least 4 ubiquitins attached, the target protein is recognized by the ubiquitin receptor in the 19S Cap of the proteasome.

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97
Q

What amino acid is always at the C-terminal end of a protein that is set to be degraded?

A

Glycine

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98
Q

Name the three enzyme families required for ubiquitination and their functions

A
  • E1: ubiquitin-activating enzymes. Create an activated E1-bound ubiquitin through a high energy thioester link
  • E2: ubiquitin-conjugating enzymes. Accept the ubiquitin from E1.
  • E3: ubiquitin ligases or E3 ligases. E3 ligases recognize the degradation signal (degron) in the protein to be ubiquitinated. E3 ligases thus confer the specificity.
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99
Q

True or False: E1 (activating enzyme) is the most specific of the ubiquitination enzymes

A

FALSE: E3 is the most specific (have over 600 different genes coding for them)

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100
Q

What is regulated degradation/regulated proteolysis?

A

Some types of E3 ubiquitin ligases regulate the mass of critical cellular proteins during different phases of the cell cycle (cyclins), development, etc

-> A type of degradation by the proteasome

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101
Q

List the three contexts that the Ubiquitin-Proteasome System Degrades Proteins

A
  1. Degradation of misfolded proteins
  2. Regulated gene expression by controlled proteolysis (ex. Control of cell cycle proteins)
  3. Hydrolysis of foreign proteins for production of antigens in the process of generating antibodies
    - -> For all of these, the specificity is primarily provided by the E3 ligases.
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102
Q

In what two ways is regulated degradation by a proteasome induced?

A

1) Activation of a ubiquitin ligase complex

2) Activation of a degradation signal on a protein

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103
Q

Give a summary of correct protein folding

A
  • Proteins begin folding co-translationally into their final conformation.
  • Molecular chaperones such as Hsp70 and Hsp60 assist in protein folding.
  • The ubiquitin ligase system marks incorrectly folded proteins and proteins whose mass is ‘regulated’ for degradation by the proteasome by polyubiquitinating the target protein.
  • Other types of ubiquitination serve other purposes in the cell such as regulation of gene expression
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104
Q

Describe how translation is down-regulated in the presence of an infection or stress

A
  • This is largely accomplished by the phosphorylation of eIF2 by protein kinases that are activated in response to the stress.

–> Reminder: when eIF2 is bound in a complex with GTP, it mediates the binding of met-tRNAi to the small ribosomal subunit. This is the first step in the initiation of translation, so the perfect step to regulate

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105
Q

What does Exchange of GDP for GTP on eIF2 Require?

A
  • Exchange of GDP for GTP on eIF2 Requires a Guanine Nucleotide Exchange Factor (GEF)
  • GDP and GTP are allosteric modulators of eIF2, and eIF2 is only active when GTP is bound to it. GDP binds very tightly to eIF2, so eIF2B, an accessory protein known as a guanine nucleotide exchange factor(GEF), is needed to get GDP off so that GTP can bind and activate eIF2.
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106
Q

What happens when When eIF2 Is Phosphorylated?

A
  • It Binds Too Tightly to eIF2B So That eIF2 Is Not Released and Cannot Bind GTP
  • Stress induces various protein kinases that can phosphorylate eIF2.
  • This mechanism is part of what puts cells into Go as protein synthesis decreases to about 20% of that in proliferating cells.
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107
Q

True or False: ~90% or eukaryotic mRNAs are translated beginning with the first AUG downstream from the 5’ Cap

A

TRUE

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108
Q

For the eukaryotic genes that are not translated beginning with the first AUG downstream from the 5’ cap, where does translation begin?

A
  • Translation of many of the remaining mRNAs begins at internal ribosome entry sites (IREs).
  • -> IREs are specialized RNA sequences of several hundred nucleotides long that fold into specific structures that bind many of the proteins that are used to initiate normal 5’ Capdependent translation.
  • -> This unusual structure allows the translational machinery to bypass the 5’ Cap structure.
  • -> Used primarily by viruses or during stress. It allows for translation under conditions where 5’ Cap-dependent translation is turned off or reduced. Helps the cell cope with stress, and it helps virus proteins get translated preferentially.
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109
Q

Compare the Initiation of Translation Using the 5’ Cap vs. IREs

A
  • IRE-initiated translation requires only a subset of the normal initiation factors and those assemble directly on the IRE rather than the Cap.
  • Some viruses produce a protease upon infection that cleaves eIF4G so it will no longer bind to eIF4E. This halts host translation. However, the clipped eIF4G will bind to IREs in the viral mRNAs, so viral proteins are preferentially made.
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110
Q

How can bacterium to adapt to rapid environmental changes?

A

Bacterial mRNAs typically have a very short half-life, usually less than ~ 2 min.

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111
Q

What is the function of an exonuclease in mRNA?

A

Exonucleases degrade mRNA from the 3’- to 5’direction, are usually responsible for mRNA degradation in bacteria

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112
Q

What is responsible for mRNA degradation in eukaryotes?

A
  • Most eukaryotic mRNAs are degraded from the 3’-end by a deadenylase and from the 5’-end after decapping by a 5’- to 3’-exonuclease.
    – Some mRNAs are internally cleaved by endonucleases. The stability of these mRNAs is specifically regulated by sequences in the mRNA
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113
Q

True or False: Degradation of Eukaryotic mRNAs Is Often Co-Translational

A

TRUE

–> As an mRNA is being translated, a deadenylase associates with the poly-A tail, shortening it over time. Once the tail is shortened to about 25 A’s, then the mRNA is decapped and exonucleases start 5’-to-3’ and 3’-to-5’ degradation

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114
Q

What are the two mechanisms of mRNA degradation in eukaryotes?

A
  • Mechanism 1: The loss of the Cap leads to rapid 5’-to-3’ degradation.
  • Mechanism 2: After the poly-A tail is reduced to about 25 A’s, there is rapid 3’-to-5’ degradation.
  • -> Mechanisms 1 and 2 can occur on the same mRNA at the same time.
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115
Q

Describe iron transport and storage in eukaryotic cells

A
  • Ferritin and the transferrin receptor are largely responsible for regulating iron levels in our bodies.
  • Ferritin binds iron within cells, stores it in the cells, and releases it as needed.
  • Transferrin carries iron in serum and when more iron is needed in cells, the transferrin receptor takes up transferrin + Fe3+ (ferric ion) and thus brings iron inside.
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116
Q

Describe Regulation of the Half-Life of Ferritin and Transferrin Receptor mRNAs by Iron Starvation (or Low Iron)

A
  • Cytosolic aconitase binds to stem loops in mRNAs that contain an iron response element (IRE) when the aconitase does not have Fe bound to it.
  • When Fe is low, aconitase binds to the IRE stem loop in the 5’end of the ferritin mRNA, and its translation is inhibited, so less Fe is stored.
  • Similarly, when Fe is low, aconitase can bind to IREs in the 3’-UTR of the transferrin receptor mRNA, stabilizing it so more transferrin receptor is made. This enables more Fe to be extracted from the serum.
117
Q

Describe Regulation of Ferritin and Transferrin Receptor mRNAs by Excess Iron

A
  • When Fe is high and binds to aconitase, the aconitase can’t bind to IREs. Thus, ferritin mRNA is translated, more ferritin is made, and more Fe is stored in cells.
  • Similarly, the Fe-bound aconitase can’t bind to IREs in transferrin receptor mRNA, so the receptor mRNA is available for cleavage. Thus, the mRNA is degraded and less Fe is taken up into the cell. The idea is to prevent too much Fe storage as it is toxic
118
Q

What is RNA interference (RNAi)?

A
  • RNAi is the inhibition of gene expression by short single-stranded RNAs.
  • These small noncoding RNAs (small ncRNAs, sncRNAs) can cause degradation of the target mRNA and/or repress its translation and/or cause heterochromatin formation.
119
Q

What do ncRNAs do?

A
  • All function to reduce gene expression by destabilizing specific RNAs, repressing their translation, or repressing transcription

–> Their mechanisms differ, however

120
Q

Give an Overview of RNA Interference in Eukaryotes

A
  • The mechanism by which silencing occurs depends on specific qualities of the RNA and the actual components of the RISC complex (called Argonaute or Piwi proteins in the figure in slides).
  • The relative contributions of these three mechanisms remain hotly debated, in part because they seem to be dependent on the particular organism.
  • The ss interfering RNA is antisense to the target mRNA
121
Q

True or False: RNA Interference is a kind of post-transcriptional gene expression

A

TRUE

  • Post-transcriptional gene expression isany control of gene expression that occurs after transcription has begun, such as regulation of the stability of an mRNA. In the case of RNAi, this is sometimes called post-transcriptional gene silencing (PTGS) because the regulation inhibitsgene expression. (However, RNAi can also affect heterochromatin formation and thus transcription.)
122
Q

What do miRNAs do?

A
  • MicroRNAs; Regulate gene expression by blocking translation of specific mRNAs and cause their degradation in lower organisms
  • In mammals, they likely destabilize specific mRNAs
123
Q

What do siRNAs do?

A
  • Small Interfering RNAs; Turn off gene expression by directing the degradation of selective mRNAs and the establishment of compact chromatin structures
124
Q

What do piRNAs do?

A
  • Piwi-interacting RNAs; Bind to piwi proteins and protect the germ line from transposable elements
125
Q

Describe miRNAs

A
  • miRNAs are endogenous ncRNAs that regulate gene expression. Thus, miRNA precursors are always genomic in origin.
  • miRNA precursors are mostly transcribed by RNA Pol II (can be Pol III) and are capped and polyadenylated just like an mRNA.
  • miRNA precursors are derived from single stranded RNA, which form a hairpin structure that is cleaved into dsRNA.
  • Complementarity to their target mRNAs is typically not perfect in animals, although it is often perfect in the seed sequence. The seed sequence is the sequence in the miRNA that recognizes the target mRNA
126
Q

True or False: miRNAs are often part of stem-loop structures

A

TRUE

–> They’re each coded on miRNA genes and the primary miRNA transcript folds back on itself, creating the stem-loop

127
Q

Describe siRNAs

A
  • siRNAs are believed to be a primitive immune response against any foreign nucleic acid molecule that can make double-stranded RNAs. These ‘foreign’ RNAs can come from within the cell (transposable elements, repeats) or outside of the cell (viruses).
  • The dsRNAs are actually the precursors for the siRNAs. (In contrast, remember, miRNAs are transcribed by Pol II or III.)
  • Because the siRNA comes directly from the ‘foreign’ RNA to be targeted, there is perfect complementarity between the siRNA and the target mRNA, which leads to mRNA degradation by endonucleolytic cleavage.
  • Many people use the terms miRNA and siRNA interchangeably. Don’t in this course!
128
Q

Describe piRNAs

A
  • Named because they interact with a subfamily of Argonaut proteins known as PIWI proteins.
  • Originally identified only in the germ-line, and the name comes from the original mutant phenotype = P-element-induced wimpy testis.
  • Long piRNA precursors are transcribed by Pol II from genomic loci known as piRNA gene clusters (10 – thousands of genes long). These clusters usually contain transposon remnants or repeats, and the piRNA precursor is transcribed antisense to the transposon. The precursor is cleaved into many piRNAs, which are single-stranded.
  • The main function of piRNAs is to silence transposition (‘jumping genes’) in the germline.
  • Recent evidence suggests piRNAs have a function in stem cells in lower eukaryotes.
  • Good evidence supporting a role for piRNAs in somatic tissues in humans is not available as yet
129
Q

Describe The Synthesis of Pre-miRNAs

A
  • miRNAs are synthesized from endogenous genes.
  • The distinguishing feature of miRNA genes is that their primary transcripts (pri-miRNAs) fold into hairpin loops.
  • Pri-miRNAs are typical RNA Pol II transcripts with a 5’-Cap and poly A tail. They can be quite long, >1000 NT.
  • The ssRNAat the 5’ and 3’ ends of the primRNA is trimmed off by a so-called Microprocessor complex, which includes the endonuclease Drosha and the RNA binding protein DGCR8. DGCR8 is needed for Drosha’s activity.
  • The pre-miRNAs are about 75 NT long.
  • This clipping occurs in the nucleus.
130
Q

Describe the four different types of miRNA genes

A

Solo miR

  • Independent transcriptional units with their own promoters, just like mRNAs
  • -> Cropped by Drosha

Clustered miR

  • Clusters of several miRNAgenes transcribed as a single pri-mRNA (uses own promoter)
  • -> Cropped, then Drosha

Intronic miR

  • miR genes can reside in introns and are transcribed with the primary transcript and spliced out later, or it can have its own promoter
  • -> Cropped, spliced, then Drosha

Conventional Mirtron

  • Mirtrons are the introns and are derived from the lariat that occurs during splicing
  • -> Very few miRNAs are mirtrons (<20)
  • -> Cropped, debranched
131
Q

Describe the processing of pre-miRNA

A
  • Drosha trims off the non-hairpin RNA.
  • Dicer is an endonuclease and cleaves off the loop region of the hairpin, leaving the ~22-23 NT ds RNA complex (cytoplasm)
  • The miRNA is loaded onto an Argonaute (AGO2)-containing complex known as RISC. RISC is a protein complex that selects a single strand of the miRNA/siRNA to target the mRNA. The sense strand is degraded because AGO2 is an endonuclease.
  • The ss miRNA binds to the target mRNA and either causes it to be degraded and/or inhibits its translation depending on the degree of complementarity and the species.
132
Q

Why is The Mechanism of Action of miRNAs in Humans Is Controversial?

A
  • Recent evidence indicates that mRNA degradation is the predominant effect in humans and other higher eukaryotes. The mRNAs are deadenylated (which prevents translation) and then degraded in P-bodies. It is unclear whether a pause in translation precedes the deadenylation or not.
  • -> P-bodies are foci in the cytoplasm involved in mRNA degradation
133
Q

Describe the Hypotheses as to How miRNAs Cause Translational Repression

A

miRNA-loaded RISC may hybridize to the 3′ UTR of an mRNA that is undergoing translation and b) block entry of ribosomal subunits at the 5′ Cap, c) inhibit transcriptional elongation, or d) promote the degradation of the mRNA by deadenylation and exonuclease activity. Other mechanisms have also been proposed such as prevention of cyclization

134
Q

How Does RISC/AGO2 Select the Right Strand?

A
  • Strand selection is thought to be dictated by the thermodynamic stability of the two strands.
  • The strand less stable at the 5’-terminus is kept as the guide strand = antisense strand.
  • This is because AGO2 binds better to the most stable strand (the passenger strand) and cleaves it, leaving the guide strand intact.
  • The little star represents where the passenger strand = sense strand is clipped by the endonuclease activity of AGO2

-> Lecture 19

135
Q

How Are Target mRNAs Selected?

A
  • miRNA/siRNA target sites were originally found in the 3’UTRs of mRNAs.
  • However, recent studies show only about 51% of the target sites are in the 3’-UTRs. Surprisingly, about 37% are actually in the coding sequence, often just upstream of the Stop codon.
  • Only a 6 – 7 NT long region at the 5’-end of the miRNA/siRNA (nucleotides 2 – 8) is necessary for recognition of the mRNA. This 6 – 7 NT is called the seed sequence.
  • miRNAs that recognize the same mRNA sequence are called seed families.
136
Q

Describe perfect an imperfect matching of seed sequences in the degradation of mRNA

A
  • Typically, the seed sequence is nucleotides 2 – 8 in the miRNA/siRNA. A perfect match is thought to lead directly to mRNA degradation by endonucleolytic cleavage. An imperfect match is thought to lead to translational repression followed by mRNA degradation in P bodies in lower eukaryotes.
  • In higher eukaryotes, the mRNAs are targeted for degradation in P bodies with an imperfect match, although there may be translational repression first
137
Q

True or False: Each mRNA May Have More Than One Target Site for a Single miRNA and May Also Have Target Sites for Several miRNA Seed Family Members

A

TRUE

138
Q

What Features Make miRNAs Especially Useful Regulators of Gene Expression?

A
  • A single type of miRNA can regulate many different target mRNAs. The targets just have to have complementarity to the miRNA.
  • Regulation by miRNAs can be combinatorial. When the bp of one miRNA fails to cleave the target mRNA, another can bind and help degrade the mRNA or assist in translational repression.
  • Occupy relatively little space in the genome considering their importance.
  • They can elicit modest effects (rheostat) rather than on/off effects
139
Q

What are Sources of siRNA?

A

Biological (‘natural’) sources
— Viruses that contain or make dsRNA
— Transposons
— Repeats that can make hairpin loops if transcribed
Experimental (research) sources
— Chemically synthesized (siRNA) that is already the correct size as it is synthesized as individual oligonucleotides and then annealed. Usually called ASOs = antisense oligomers
— Insertion of the siRNA sequence into a retroviral plasmid. The RNA made from this is called shRNA (short hairpin) because it is designed to form a short hairpin just like pre-miRNAs and is processed by Dicer

140
Q

Describe dsRNA in viruses

A
  • Most RNA viruses produce dsRNAs as intermediates or products of genomic replication.
  • Many DNA viruses generate dsRNA from convergent (antisense) transcription of their genome.
  • Most viruses contain dsRNA or make dsRNA upon infection or replication!
  • –> Because the siRNAs are made directly from the template (not from a separate gene like the miRNAs), there is very high complementarity between the siRNA and the target RNA. Thus, either strand of the siRNA can work to interfere with the virus.
141
Q

Describe the basic steps of initiation of RNAi (generation of the mature miRNA or siRNA)

A
  • The double stranded pre-miRNA or pre-siRNA is cleaved in the cytoplasm into small dsRNA pieces of about 22 NT with a 3’ overhang by a Dicer complex
  • Risc binds to the dsRNA molecule and selects a strand to use. For an miRNA, it is antisense to mRNA. For an siRNA, selection seems to be mostly random. The selected strand is known as the guide strand. This occurs in the cytoplasm.
142
Q

Describe the basic steps of execution of RNAi (mRNA degradation or inhibition of translation)

A
  • The guide strand-Risc complex binds to target mRNA molecules with a complementary sequence through base pairing
    – The targeted mRNA molecule is then:
    — Endonucleolytically cleaved by Ago2 in the case of siRNAs and degraded
    — Deadenylated and degraded (exonuclease degradation) in P-bodies (miRNAs in mammals)
    — Blocked for translation by the bound complex, and then degraded in P-bodies (miRNAs in plants, lower eukaryotes, & mammals)
143
Q

Describe Biogenesis of piRNAs

A
  • piRNAs are the most common of the sncRNAs, with hundreds of thousands of different ones, expressed mostly in the germline
  • The genes are grouped in clusters and are often bidirectional in a particular region.
  • The genes are found everywhere: in transposons, protein-encoding genes, and intergenic loci
  • These clusters can be a million nucleotides long
144
Q

Compare the Biogenesis of piRNAs to miRNAs and siRNAs

A
  • piRNAs are derived from single-stranded RNAs and are processed very differently from miRNAs and siRNAs.
  • piRNA processing is Drosha- and Dicerindependent.
  • An endonuclease(s) clips the precursor pi RNA into fragments. To prevent degradation, the ends are modified.
  • There is an amplification step to increase the amount of piRNA made from the single precursor RNA through a mechanism known as the ping pong mechanism.
145
Q

Describe piRNA Biogenesis in Drosophila

A

An unknown endonuclease clips the piRNAprecursor into fragments. These piRNAs go into a perinuclear ‘body’ called a Yb body and are bound with PIWI proteins. The endonuclease Zuc (Zucchini) creates the mature piRNA, which has a U at the 5’-end. This piRNA-PIWI protein complex can go back to the nucleus and has epigenetic effects to silence chromatin. (It can also mediate mRNA decay and perhaps translational inhibition, not shown here.)

146
Q

What is RITS?

A
  • RITS = RNA induced transcriptional silencing protein complex
  • The RITS complex attracts proteins that modify histones and DNA, thus repressing transcription (in Drosophilia).
  • This has not been demonstrated in mammals except for piRNAs.
147
Q

Give a Summary of The Effectors and Types of RNAi

A
  • All mechanisms lead to a decrease in a specific RNA or protein.
  • Degradation of mRNA via miRNAs is by exonucleases after removal of the Cap and polyA tail, and it occurs in P bodies. Degradation of RNA via siRNAs is by the endonuclease AGO2 and occurs in the cytoplasm.
148
Q

Describe Addition of exogenous RNA experimentally

A

This is an experimental tool. The siRNA is already the correct length and can be designed to be perfect or imperfect so that it stimulates mRNA degradation and/or inhibition of translation.

149
Q

Describe viral infection as it relates to RNAi

A

At some point, dsRNA is made. It might need to be processed by Drosha. The dsRNA is trimmed to the correct size by Dicer. The match may be perfect or imperfect so that it stimulates mRNA degradation and/or inhibition of translation.

150
Q

Describe transposon RNA as it relates to RNAi

A

RNA synthesized from an RNA or DNA transposon can become doublestranded. The dsRNA is trimmed to the correct size by Dicer. The match may be perfect or imperfect so that it stimulates mRNA degradation and/ or inhibition of translation.

151
Q

Describe miRNAs (summary)

A

synthesized endogenously from many miRNA genes. The RNA folds back on itself to become double-stranded and is called a pri-miRNA. This can be very long. Drosha trims the pri-miRNA in the nucleus and the resulting~75 NT pre-miRNA is shuttled to the cytoplasm. The match is usually imperfect, leading to mRNA degradation in P-bodies (in mammals) and/or translational repression. This is the most important natural form of RNAi.

152
Q

Describe Inhibition of transcription and chromatin condensation in RNAi

A

attachment of RITS-si/miRNA complex to an mRNA can lead to histone acetylation and chromatin condensation, at least in yeast and plants and with piRNAs.

153
Q

Describe addition of shRNA experimentally

A

An shRNA (presiRNA) sequence is inserted into a retroviral plasmid and that plasmid is inserted in a retrovirus. When the virus infects cells, the shRNA is made. The shRNA is processed as in miRNAs

154
Q

What Is the Biological Impact of miRNAs?

A
  • Knock out studies suggest that many conserved miRNAs are dispensable for animal development and viability, and few have an obvious effect. So are they really important? Evidence says yes:
  • – They comprise 1- 2% of all genes in worms, flies, and mammals.
  • – Because of the large number of targets, it’s thought that most genes are under their control.
  • – miRNAs and their targets often exhibit striking evolutionary conservation.
  • – Animals carrying mutations that impair miRNA processing are not viable, indicating complete loss of miRNA activity is not compatible with life.
155
Q

Why are miRNAs Are Thought to Act Primarily as Rheostats of Transcriptional Programs?

A
  • miRNAs do not appear to act as master regulators of gene expression, unlike transcription factors.
  • Instead, they act to fine-tune transcriptional programs, as components of complex signaling networks, and to buffer the effects of the environment and genetic variability.
  • However, major effects can be seen with over or underexpression of a specific miRNA if its targets are functionally linked in a pathway.
  • – Ex: in mice, deletion of miR-128 causes fatal epilepsy due to derepression of several components in the MAPK pathway, which inappropriately activates it.
156
Q

True or False: miRNAs Regulate Many Cellular Processes and Their Abnormal Expression Is Linked to Disease

A

TRUE

157
Q

Describe miRNA and Cancer: Example let - 7 miR

A
  • Let-7 miRNA is processed from its long dsRNA precursor by Drosha and then by Dicer.
  • Mature let-7 miRNA is known to inhibit the expression of oncogenes such as Ras, Myc, cyclin D, Cdc25a.
  • Oncogenes when expressed at high levels can transform a normal cell to proliferative cancer cells.
  • Decreased levels of miRNA can lead to increased expression of oncogenes and cancer.
  • Aberrant miRNA levels are observed in many different types of cancer.
158
Q

Why is there Considerable Interest in Using RNAi Experimentally and Therapeutically ?

A
  • Gene Function Analysis: RNAi allows scientists to knock down expression of a targeted gene and observe the effects of partial to full loss of function.
  • Can also be used, of course, to study the functions of specific miRNAs as well as proteins
  • Can use siRNAs directly (cells, C. elegans) or shRNAs (whole animals)
  • shRNAs are more stable and produce more sustainable effects
159
Q

What is an miRNA replacement strategy in RNAi therapy?

A

seek to mimic the functions of specific miRNAs by delivering small dsRNAs

160
Q

What is an miRNA targeting strategy in RNAi therapy?

A

antisense oligomers (ASO) are in development to target specific miRNAs so they can’t bind to their target mRNAs

161
Q

Describe Issues Associated with RNA-Based Therapies

A

– Each miRNA has dozens to hundreds of target mRNAs so there are off target effects. However, these effects are often mild, and generally not lethal.
– Because of seed families, knockout of any one member of a seed family rarely has any phenotype, although sometimes the phenotype is observed under a particular type of stress.
– ASOs are substrates for serum nucleases so they are unstable, and they are unable to penetrate the cell membrane. They also have poor tissue penetration when delivered systemically.
–» Some of these have been overcome by using modified oligos or liposomes/nanoparticles for delivery.

162
Q

What is the definition of cellular communication?

A

Cellular communication is the generation, transmission, reception, and processing of mostlyextracellular information

163
Q

Definition: cell signaling

A

Cell signaling refers to the biochemical mechanisms by which cells receive information from another cell or from the environment and utilize this information to cause a change in cell function

  • Most signaling and communication between cells involves chemical signals (e.g., peptides, steroids, fatty acids, amino acid derivatives).
  • -> However, there are environmental signals (mostly sensory) such as light, sound, heat, taste, and pressure that are essential to physiological function
164
Q

Definition: Signal transduction

A

The conversion of information from one physical or chemical form to another

165
Q

Definition: Agonist

A

A ligand (signal) that activates a receptor

166
Q

Definition: Antagonist

A

A ligand that blocks the actions of the agonist by competitively binding to the receptor

167
Q

Definition: Desensitization (as it refers to signal transduction)

A

Inactivation of the receptor or its signaling pathway

168
Q

Why is Understanding Signaling Pathways Challenging?

A
  • Cells employ hundreds of distinct signaling pathways involving hundreds to thousands of proteins.
  • The signals are transient so are not easily monitored.
  • Most pathways have positive and/or negative feedback loops, which contribute to the transience of the signal.
  • Most pathways branch and converge multiple times.
  • The response of pathways often depends on the strength and the temporal pattern of the stimulus.
169
Q

Definition: Proto-oncogene

A

a normal cellular gene that promotes cell growth, survival, and/or proliferation as part of its normal function

170
Q

Definition: oncogene

A

a cellular gene that is cancer-promoting (onco = mass) due to mutations or dysregulation (to be discussed later). Originally a proto-oncogene.

171
Q

Definition: tumor-suppressor gene

A

a gene that opposes cell proliferation and is growth inhibiting and/or involved in repairing the genome

172
Q

True or False: Studying Retroviruses Illuminated Steps in Many Signaling Pathways

A

True

173
Q

List all of the signals in signal transduction

A
• Antigens 
• Cell surface glycoproteins, extracellular matrix 
• Developmental signals/gradients 
• Growth factors 
• Hormones 
– Peptide/protein (insulin, glucagon)
 – Steroid 
– Amines (epinephrine, norepinephrine, thyroxine)
 • Environmental 
– Light 
– Mechanical touch/pressure/stretch
 – Odorants, Pheromones 
– Sound
 – Tastants 
• Neurotransmitters 
• Metabolites (fatty acids, bile acids)
174
Q

How do hydrophilic ligands work?

A
  • Can’t pass through the membrane (so the receptors are on the outside of the membrane)
  • -> Proteins or polypeptides
  • -> Derivatives of amino acids: epinephrine, norepinephrine
  • Ligands are stored
  • Rapid response: seconds to minutes (primarily at the level of enzyme activity)
  • Cell-surface receptors
175
Q

How do hydrophobic ligands work?

A
  • Can pass through the membrane (So, the receptors are INSIDE the cell)
  • -> Derivatives of cholesterol: steroid hormones, Vit D
  • -> Derivatives of fatty acids: retinoids, eicosanoids
  • Ligands are NOT stored
  • Slow response: minutes to hours (primarily at the level of transcriptional activity)
  • Intracellular receptors
  • Generally transported by carrier proteins from which the dissociate before entrance
176
Q

What are the General Features of Receptors?

A
  • Target cells respond to a signal by means of receptor proteins
  • Receptors bind the signal and initiate a response.
  • Receptors have multiple functional domains (ex. ligand binding, response).
  • Each cell has many different kinds of receptors.
  • Each cell has a characteristic number of each kind of receptor.
  • The binding of ligand to receptor resembles typical Michaelis-Menton kinetics.
177
Q

What do Ion-channel-coupled receptors (transmitter-gated ion channels) do?

A

Ion-channel-coupled receptors (transmitter-gated ion channels) open or close specific ion-gated channels in response to binding of the signal molecule, which is typically a neurotransmitter. (see pages 627-631).
Ex: acetylcholine nicotinic receptor

178
Q

What do G protein-coupled receptors (GPCRs) do?

A

G protein-coupled receptors (GPCRs) interact with G (guanine nucleotide binding) proteins in the presence of the signal molecule to promote GDP/GTP exchange. The active G protein activates a membrane enzyme and/or ion channel.
Ex: Glucagon, sensory receptors

179
Q

What do Enzyme-coupled receptors do?

A

Enzyme-coupled receptors have intrinsic enzymatic activity in their cytoplasmic domain or are tightly associated with an enzyme. In either case, enzyme activity is activated by binding of the signal molecule to the receptor. Examples: receptors for insulin, growth hormone, growth factors, cytokines

180
Q

Describe Contact-Dependent signaling

A
  • In contact-dependent signaling, the signal molecule is displayed on the cell surface and consequently can only influence with direct membrane to membrane contact.
  • The specificity comes from the receptor but also from the proximity
181
Q

Describe Paracrine signaling

A
  • Local mediators (ex. cytokines) are secreted into the extracellular space by one cell type and act locally on/in a differentnearby cell type.
  • The specificity lies with the receptor but also with the proximity as many of the local mediators have a very short half-life
182
Q

Describe autocrine signaling

A

Autocrine signaling is a specialized form of paracrine signaling. It involves local mediators that act on the same cell that released the signal molecule. Ex: Cancer

183
Q

Describe synaptic signaling

A
  • Neurons release neurotransmitters at synapses, which are often located far from the cell body. However, this could be considered a specialized form of paracrine signaling as the target cell is in close proximity to the terminus of the axon.
  • Specificity comes from the receptor and also from one-to-one communication between the axon of the signaling neuron and the target cell
184
Q

Describe endocrine signaling

A

• Hormones are secreted by endocrine glands into the blood and consequently can affect any cell or tissue in the body at great distances from the endocrine cell. •The specificity lies with the receptor and with the location of the receptor, i.e. not all cells have all receptors

185
Q

How Do the Signal and the Receptor Hook Up?

A
  1. Extracellular Signals Can Act Over Short or Long Distances
  2. Each Cell Depends on Many Extracellular Signals to Function Properly
  3. Different Cells Can Respond Very Differently to the Same Signal Molecule
186
Q

What steps must occur for cell signaling to happen (dont need to know the order)?

A
  1. Synthesis of the signal (if endogenous)
  2. Release of the signal
  3. Transport to the target cell
  4. Binding to a specific receptor protein
  5. The steps involved in eliciting the response to the signal
  6. Removal of the signal
  7. Termination of the response
187
Q

What is Specificity is determined by in cell signaling?

A

– complementarity between the signal and the receptor, i.e. the signal molecule fits the binding site in its receptor
– the tissue- and cell-specific distribution of receptors
– the tissue- and cell-specific distribution of the intracellular response systems
– proximity of the signal

188
Q

Describe Amplification of cell signaling

A

The signal of ligand binding is sometimes geometrically increased as a result of an enzyme cascade

  • -> Increases the intensity of the signal
  • -> Prolongs the duration of the signal
189
Q

Describe the mechanisms of Desensitization (Adaptation) of Receptors

A
  1. Receptor sequestration: The receptor is internalized into an endosome but often recycles back to the membrane once the amount of the signal molecule has dropped.
  2. Receptor down-regulation: The receptor is internalized into an endosome, trafficked to a lysosome, and then degraded. New synthesis is required to restore receptor levels.
  3. Receptor inactivation:The receptor is inactivated, often by modification such as by phosphorylation or dephosphorylation.
  4. Inactivation of an intracellular signaling protein: A signaling protein closely associated with the receptor is inactivated.
  5. Production/activation of an inhibitory protein: An inhibitory protein prevents the receptor from signaling even though signal is there.
190
Q

What does cAMP do?

A

Activates protein kinase A (PKA)

191
Q

What does cGMP do?

A

Activates protein kinase G (PKG) and opens cation channels in rod cells

192
Q

What does DAG do?

A

Activates protein kinase C (PKC)

193
Q

What does IP3 do?

A

Opens Ca2+ channels in the endoplasmic reticulum

194
Q

What purposes do Intracellular Relay Systems Serve?

A
  • Transmit the extracellular signal to the correct cellular compartment (ex. membrane to nucleus)
  • Transduce the signal to the correct form
  • Amplify the signal
  • Integrate the signal with other pathways
  • Diversify the signal to multiple pathways/compartments
  • Provide specificity
  • Increase the speed and efficiency of the response
195
Q

True or False: A Sequence of Two Inhibitory Signals Produces a Positive Signal

A

TRUE

–> It is a double negative

196
Q

How Are the Proteins in the Relays/Cascades Regulated?

A

• Covalent modification by phosphorylation or dephosphorylation
– Addition of a phosphate group with its 2 negative charges can change the conformation of the protein, which can affect protein activity
– The phosphate group can serve as a recognition signal
– The phosphate group can mask a binding site and thus disrupt protein-protein interactions
– Cellular localization
• Allosteric modification
- Guanine nucleotide (GTP/GDP) binding

197
Q

Describe Protein Phosphorylation/Dephosphorylation

A

• Protein kinases catalyze the transfer of the γ-phosphoryl from ATP to a hydroxyl group on serine (84% in mammals), threonine (15%), or tyrosines (<1%). There are two major groups of protein kinases:
–> Serine/threonine protein kinases put phosphates on serine or threonines.
–> Tyrosine protein kinases put phosphates on tyrosines
• Protein phosphatases remove the phosphates

198
Q

How can Protein Phosphorylation Can Lead to Either Activation OR Inactivation?

A

There is no generalization as to whether a protein will be activated or inactivated by phosphorylation. Keep in mind that phosphorylation or dephosphorylation may lead to only partial activation or inactivation, i.e. this is not necessarily an on/off switch mechanism as shown here. Sometimes you need multiple phosphorylation events to fully activate or inactivate a protein.

199
Q

Give some general features of Protein Kinases

A

• All protein kinases share a conserved catalytic core of ~290 amino acids that include the active site and ATP binding sites.
• The catalytic mechanism used by all protein kinases is similar.
• These observations suggest that all protein kinases evolved from a common ancestral protein kinase gene.
Each protein kinase is regulated by different signals and in turn recognizes and phosphorylates a different set of target proteins at a highly specific site(s).
• Some protein kinases are highly specific and only phosphorylate a single substrate; most are more general and modify many proteins

200
Q

What does GAP (GTPase activating proteins) do?

A

speed up protein inactivation by promoting the hydrolysis of GTP.

201
Q

What does GEF (guanine exchange factors) do?

A

speed up protein activation by promoting the exchange of the GDP for GTP

202
Q

What are the main components of the GPCR membrane complex?

A
  • Receptors
  • G proteins
  • Effectors
203
Q

Give examples of some effectors

A
  • Adenylyl cyclase
  • Phospholipase C-β
    •Ion channels
    •cGMPphosphodiesterase (transducin)
204
Q

True or False: Drugs aimed at specific GPCRs are among the most effective and commonly-used pharmaceuticals

A

TRUE

205
Q

Describe the GPCR conformation

A
  • All GPCRs Adopt a Similar Conformation of Seven Transmembrane Domains
  • Transmembrane domains are 24-25 aa long, hydrophobic α-helical segments
  • C terminus on inside of cell, N terminus on outside
206
Q

What are G proteins?

A

G Proteins Are a Family of Signaling Proteins that Bind Guanine Nucleotides and Function as Molecular Switches

207
Q

True or False: G proteins are active when bound to GDP

A

FALSE: They are INACTIVE when bound to GDP

–> Active when bound to GTP

208
Q

Describe the alpha subunit of G proteins

A

The α subunit is a GTPase. It contains 2 domains: a Ras domain that is similar to other GTPases and forms one face of the binding pocket and an AH (α helical) domain that forms the other side of the guanine nucleotide binding pocket

209
Q

What is Isoprenoid lipid moiety?

A
  • Holds the subunits of G proteins together
  • -> G proteins remain associated with the membrane and close to the GPCRs. This increases speed and probably specificity. They are peripheral membrane proteins
210
Q

What does Gs do?

A

Activates adenylyl cyclase, activates Ca2+ channels

211
Q

What does Gi do?

A

Inhibits adenylyl cyclase and activates K+ channels

212
Q

What does Go do?

A
  • Activates K+ channels, inactivates Ca2+ channels

- Activates phosholipase C-beta

213
Q

What does Gq do?

A

Activates phospholipase C-beta

214
Q

How does binding of ligand to receptor activate G proteins?

A

Change of cis to trans conformatons in response to environmental changes

–> Ex: Rhodopsin changes in response to light

215
Q

Describe Activation of a G protein by an Activated GPCR

A
  1. Binding of ligand to the GPCR causes a conformational change in the receptor that allows the G protein to bind to it.
  2. Binding of the G protein to the activated GPCR alters the conformation of the G protein and the AH domain of the α subunit moves out, allowing GDP to escape.
  3. GTP binding to the α subunit closes the nucleotide binding pocket, which causes conformational changes such that the βγ subunit dissociates from the α subunit
  4. The freed α subunit and the βγ subunit are now able to regulate downstream effector molecules (not shown).
  5. The GPCR stays active as long as ligand is bound so it can activate many G proteins.
216
Q

What does Desensitization of GPCRs Involve?

A

Desensitization of GPCRs Involves Phosphorylation by GRKs (G-Protein Receptor Kinases) and Interaction with Arrestin

  • -> Arrestin is going to prolong desensitization by preventing dephosphorylation of the receptor
  • -> beta -ARK = beta-adrenergic receptor kinase
217
Q

What are the three steps of GPCR desensitization?

A
  1. Receptor inactivation by phosphorylation by GRK
  2. Binding of arrestin to the phosphorylated receptor
  3. Receptor sequestration or down-regulation if the signal remains high for very long
218
Q

True or False: cAMP is very stable

A

FALSE: cAMP is very unstable as it is hydrolyzed by specific phosphodiesterases (PDEs) to form 5’-AMP

219
Q

How does adenylyl cyclase synthesize cAMP?

A

Adenylyl cyclase synthesizes cAMP from ATP in a cyclization reaction that is extremely rapid

220
Q

What is the effect in muscle of an increase in cAMP levels?

A

Adrenaline is stimulated and the effect is glycogen breakdown

221
Q

What is the effect in liver of an increase in cAMP levels?

A

Glucagon is stimulated and the effect is glycogen breakdown

222
Q

What is the effect in fat of an increase in cAMP levels?

A

Adrenaline, ACTH, glucagon, and TSH are stimulated and the effect is triglyceride breakdown

223
Q

Describe how cAMP Is the Allosteric Activator of Protein Kinase A

A
  • Protein kinase A = PKA = cAMP-dependent protein kinase
  • PKA is a serine/threonine protein kinase.
  • The exact targets are cell type-dependent.
  • In the absence of cAMP, the C (catalytic) subunits are bound to R (regulatory) subunits, which inhibits the catalytic activity.
  • Binding of cAMP to the R subunits, allows C to be free and active
224
Q

Describe how PKA Is Localized within the Cell by Binding to ‘Anchoring’ Proteins

A
  • A family of A-kinase anchoring proteins (AKAPs) bind to the R subunit to localize PKA to specific subcellular sites (e.g., nuclear envelope, plasma membrane, cytoskeleton)
  • Different cells express different PKA anchoring proteins, allowing cell-specific effects of cAMP to occur
225
Q

True or False: The Adenylyl Cyclase Cascade Can Trigger Changes in Transcription as well as in Enzyme Activity

A

TRUE

–> Phosphorylation of many cellular proteins is the major response to activation of PKA!!

226
Q

True or False: It is easy to predict whether or not a particular serine or threonine is phosphorylated by PKA.

A

FALSE: Because of the degree of flexibility in the consensus site, it is NOT easy to predict whether or not a particular serine or threonine is phosphorylated by PKA.

–>This is typically done experimentally.

227
Q

Describe Glucagon Signaling as an Example of an Adenylyl Cyclase Cascade

A
  • Blood glucose levels in humans are homeostatically maintained at a level of 5.5 mM; certain tissues (e.g., your brain) don’t do well if levels fall much below this level.
  • Glucagon is a polypeptide hormone produced in a signaling cell (the α cell in endocrine pancreas) that is secreted in response to low blood glucose levels (stimulus).
  • Glucagon circulates in blood until it reaches its specific receptor on the cell surface of liver cells.
  • -> Glucagon Is Not Released When Blood Glucose Levels Are High
  • -> Binding of Glucagon to its Receptor Activates Gs, Which Activates Adenylyl Cyclase
228
Q

True or False: Adenylyl cyclase always has some basal activity. Gi inhibits that basal activity in response to ligand binding to its receptor.

A

TRUE

229
Q

True or False: Cholera Is Caused by the Irreversible Activation of Gs by ADP-Ribosylation

A

TRUE

–> ADP-Riboslylation of Gs Inhibits Its GTPase Activity so Gs Constantly Activates Adenylyl Cyclase

–> A similar ADP-ribosylation of Gi by pertussis toxin prevents Gi from interacting with its receptors, and cAMP levels are not repressed.

230
Q

Describe how Both Cholera Toxin and Pertussis Toxin Cause Disease by Inappropriately Increasing cAMP Levels

A

• ADP-ribosylation of Gs inhibits its GTPase activity, meaning that Gs is permanently in its GTP-bound state and is thus active.
— This leads to increased cAMP levels and increased PKA activity
— This leads to increased loss of ions (Na+, K+, HCO3-) and water into the intestinal lumen
• ADP-ribosylation of Gi prevents it from interacting with its ligand-bound receptors, so Gi remains in the GDP-bound state and is thus inactive.
— So, Gi cannot inhibit the basal activity of adenylyl cyclase.
— This leads to increased cAMP and increased PKA activity.

231
Q

What does The Trimeric G Protein Gq do?

A
  • Activates Phospholipase C-β
  • Phospholipases are a family of enzymes that hydrolyze membrane phospholipids at different positions in the molecule.
  • Phospholipases of the C subgroup cleave the phosphodiester bond between the glycerol backbone and the phosphate head group.
232
Q

True or False: Activated Phospholipase C-β Cleaves the Membrane Lipid Phosphatidylinositol 4,5-bisphosphate

A
  • Phosphatidylinositol (PIP2) is an uncommon membrane phospholipid found on the cytosolic face of the plasma membrane. It is the starting point for several second messengers.
233
Q

List the Major Steps in Signaling Through the PI Cascade

A
  • The ligand binds to the GPCR; conformational changes in the GPCR activate Gq
  • The activated Gq activates phospholipase C-β (PLC-β)
  • PLC-β hydrolyzes phosphoinositol 4,5-bisphosphate (PIP2) to diacylglycerol (DAG) and inositol 1, 4, 5-trisphosphate (IP3)
  • DAG helps activate protein kinase C (PKC)
  • IP3 binds to IP3-gated Ca2+ channels (IP3 receptors) in the endoplasmic reticulum outer membrane, releasing Ca2+ into the cytoplasm.
  • The Ca2+ helps activate PKC
  • PKC phosphorylates target proteins, many of which are involved in cell proliferation
  • Ca2+ and DAG have other signaling roles as well
234
Q

Describe The Protein Kinase C Family

A
  • PKCs are recruited to the plasma membrane by binding to the second messenger DAG (diacylglycerol). • Many PKCs are also activated by Ca2+.
  • The biological importance of PKCs was shown years ago by the fact they are targets of phorbol esters (TPA), plant compounds that promote tumor formation (tumor promoters).
  • Phorbol esters mimic the structure of DAG but are stable and hence keep PKC active for an extended time. This causes tumors.
  • Among their other roles, PKCs increase growth factor signaling and thus cell proliferation
235
Q

What is/are the second messenger(s) in the phosphoinositide pathway?

A

DAG (diacylglycerol) and Ca2+ (calcium) and IP3 (a ligand)

236
Q

What are the Roles of DAG in Signaling?

A
  1. DAG helps activate some PKC family members
  2. DAG is the precursor for arachidonic acid and eicosanoids such as prostaglandins (PGs) – PG-Es regulate many important functions such as pain, vasodilation, suppression of gastric acid release, platelet activation, uterine contraction, inflammation
237
Q

What are the Roles of Calcium in Signaling?

A
  • Cytoplasmic Ca2+ is kept low (<0.2 μM, ~10-7 M) even in the face of high extracellular Ca2+ (2 mM, ~10-3 M) by ion pumps so that it doesn’t form a salt with the cytoplasmic phosphate.
  • External stimuli can induce a transient increase in cytoplasmic Ca2+ by opening ion channels in the plasma membrane or endoplasmic reticulum.
238
Q

Describe how Calmodulin (CaM) Is a Major Mediator of Ca2+ Signaling

A
  • Calmodulin (CaM) is ubiquitous and abundant (1% of cellular protein).
  • When CaM has at least 2 Ca2+ bound, it can change conformation in an almost a switch-like manner.
  • This change in conformation enables CaM to bind to other proteins (shown here in red). The binding of CaM helps activate those proteins.
  • CaM has no enzymatic activity but functions as a subunit of many enzymes. Thus, it is an allosteric effector.
239
Q

What proteins can Ca2+ and CaM Allosterically Modulate?

A
  • Ca/CaM protein kinases
  • eNOS, nNOS (nitric oxide synthase)
  • PKC
  • Phosphorylase B kinase
240
Q

Describe how Phosphorylase B Kinase Is an Example of a CaM-Activated Protein

A
  • Ca2+ and cAMP/PKA crosstalk to modulate certain targets such as glycogen phosphorylase kinase
  • 2 subunits of phosphorylase kinase are substrates for PKA
  • Phosphorylation of these lowers the [Ca2+] needed for activation and increases the Vmax of the enzyme.
  • Thus, cAMP and Ca2+ crosstalk to stimulate glycogen breakdown in muscle.
241
Q

True or False: Enzyme-Coupled Receptors Are Either Enzymes or Are Very Closely Associated with Enzymes

A

TRUE

242
Q

How do Enzyme-coupled receptors differ from G protein coupled receptors?

A

Enzyme-coupled receptors:
– have only one transmembrane domain per receptor monomer (except insulin receptor)
– do not interact with G proteins, but instead use enzymatic activity to transduce the signal of ligand binding

  • -> Most of the receptors in this class are protein kinases; however, some receptors have protein phosphatase or guanylyl cyclase activity
  • -> In most cases, activation of enzyme activity occurs in response to dimerization of receptor monomers upon ligand binding
243
Q

What do Receptor tyrosine kinases (RTKs) do?

A

phosphorylate proteins on specific tyrosines

244
Q

What do Cytokine receptors (tyrosine kinase-associated receptors) do?

A

have no intrinsic enzymatic activity but associate with intracellular tyrosine kinases (An internal enzyme)

245
Q

What do Receptor serine/threonine kinases do?

A

phosphorylate proteins on specific serines or threonines

–> TGFbeta receptor is one of these

246
Q

What do Receptor guanylyl cyclases do?

A

synthesize cGMP

–> Important for vasodilation

247
Q

What do Receptor tyrosine phosphatases do?

A

remove phosphoryls from tyrosines

–> Not much known about these– not how they’re activated or what their ligands are

248
Q

Describe Receptor Tyrosine Kinases (RTKs)

A
  • RTKs are the most abundant type of enzyme-coupled receptor, with about 60 human members classified into 20 structural families.
  • In all cases, binding of an ligand activates the cytosolic internal tyrosine kinase domain
  • RTKs regulate cell proliferation, cell growth, cell differentiation, cell migration, and cell fate during development
  • RTKs were discovered by studying a class of signal molecules known as growth factors (e.g., Epidermal Growth Factor = EGF, Fibroblast Growth Factor = FGF)
  • Growth factors are extracellular protein signaling molecules that act primarily as local mediators to stimulate growth and proliferation of specific cell types
249
Q

True or False: RTKs Typically Span the Plasma Membrane Once and Are Monomers Without ligan

A

TRUE

–> Has a dimerization domain so that it can become activated by binding to another monomer that has ligand bound

250
Q

What does epidermal growth factor (EGF) do?

A

Stimulates cell survival, growth, proliferation, or differentiation of various cell types; acts as an inductive signal in development

–>EGF receptors

251
Q

What does insulin do?

A

Stimulates carbohydrate utilization and protein synthesis

–>Insulin receptor

252
Q

What does insulin-like growth factor (IGF1) do?

A

Stimulates cell growth and survivial in many cell types

–>IGF receptor 1

253
Q

What does nerve growth factor (NGF) do?

A

Stimulates survival and growth of some neurons

–> Trk receptors

254
Q

What does platelet-derived growth factor (PDGF) do?

A

Stimulates survival, growth, proliferation, and migration of various cell types

–> PDGF receptors

255
Q

What does Macrophage-colony-stimulating factor (MCSF) do?

A

Stimulates monocyte/macrophage proliferation and differentiation

–> MCSF receptor

256
Q

What does fibroblast growth factor (FGF) do?

A

Stimulates proliferation of various cell types; inhibits differentiation of some precursor cells; acts as inductive signal in development

–> FGF receptors

257
Q

What does vascular endothelial growth factor (VEGF) do?

A

Stimulates angiogenesis

–> VEGF receptors

258
Q

What does ephrin do?

A

Stimulates angiogenesis; guides cell and axon migration

–> Eph receptors

259
Q

True or False: In the inactive or basal state, RTKs are monomers anchored in the plasma membrane by multiple transmembrane domains

A

FALSE: In the inactive or basal state, RTKs are monomers anchored in the plasma membrane by a SINGLE transmembrane domain

–> except insulin receptor

260
Q

Describe how RTKs Contain an Intracellular Tyrosine Kinase Domain

A
  • The intracellular domain of RTKs contains a ligandactivated tyrosine kinase domain
  • -> The receptors themselves are the initial substrates of that tyrosine kinase activity
  • Binding of ligand causes receptor monomers to dimerize in the plasma membrane, which activates the tyrosine kinase activity of one or both monomers
261
Q

True or False: Dimerization of RTKs Causes Activation of the Intracellular Tyrosine Kinase Activity

A

TRUE

–> Ligand binding causes two RTK monomers to come together and they phosphorylate each other (transphosphorylation)

262
Q

Describe how Once the Internal Domains of the RTK Are Phosphorylated, They Serve as Docking Sites for Signaling Proteins

A
  1. The ligand can be a dimer itself and thus bind to the two receptors simultaneously
  2. A monomeric ligand can bind to 2 receptors simultaneously to bring them together.
  3. Each receptor can bind a ligand, and then the receptors dimerize.
  4. Some receptors like the insulin receptor are already a “dimer”, and the ligand just induces a conformational that activates the kinase activity.
263
Q

Which type of protein domain(s) recognize(s) phosphotyrosines?

A

SH2 and PTB

264
Q

True or False: Different phosphorylated tyrosines are recognized by different signaling molecules

A

TRUE

265
Q

Describe how Some Proteins That Are Recruited to the Activated RTK Serve as ‘Adaptors’

A
  • Some proteins recruited to activated RTKs have no enzymatic activity and instead act as adaptor (or bridging) proteins that link intracellular signaling proteins together
  • One of the first adaptor proteins, which was identified by its ability to bind to RTKs, was Grb2. Grb2 typically links the MAP kinase cascade to RTKs.
  • Grb2 is composed of little more than a central SH2 domain surrounded by two SH3 domains
  • The SH3 domain (src homology domain 3) is a distinct modular interaction domain that recognizes proline-rich peptides 9-10 residues long in proteins
266
Q

True or False: Adaptor Proteins Link or Bridge Two Signaling Proteins Together Through Interaction Domains Such as SH3 Domains

A

TRUE

267
Q

True or False: RTKs are often mutated structurally in cancer

A

FALSE: They are often mutated via OVEREXPRESSION in cancer, NOT structurally

–> RTKs are proto-oncogenes because their primary function is to cause cell proliferation

268
Q

Why are RTK’s Proto-Oncogenes ?

A

i) The RTK can become mutated so that it dimerizes and becomes constitutively active without the ligand
ii) the external domain can be truncated, which also activates the receptor without ligand.

269
Q

True or False: Mutation of the Her2 and EGF Receptors Leads to Activation Without Ligand

A

TRUE

-> Her2/Neu is often mutated or overexpressed in “hormone-independent” breast cancer (BCa).

270
Q

List the four major pathways that are activated by RTKs

A

i) Src
ii) PI
iii) PI 3-kinase
iv) MAP kinase

271
Q

Describe The Src Tyrosine Protein Kinase

A
  • Functions primarily in cell migration
    • Src was the first protein kinase discovered because it was part of the Rous sarcoma virus.
    • It is a member of a subfamily of 9 similar protein kinases that are only found in multicellular animals.
    • These are all cytosolic tyrosine kinases, although covalent binding to a fatty acid attaches them to the plasma membrane
272
Q

Give A Summary of Signaling Through the Src Tyrosine Kinase

A

After Src is activated, it phosphorylates the RTK on other sites, which augments receptor kinase activity and creates additional SH2 recruitment sites. This allows the binding of Grb2-Sos and PI 3-kinase. In other words, activation of pathways is somewhat sequential.

273
Q

Describe Src as an Oncoprotein

A
  • Src is a key protein in promoting cell survival, cell proliferation, cell migration, and angiogenesis.
  • These are all hallmarks of cancer, especially metastatic cancer, and 50% of tumors from colon, liver, lung, breast, and the pancreas have abnormally activated Src.
  • A number of small molecules are in clinical use (desatinib = Sprycel™ for CML) and others are in clinical trials that inhibit Src. These include competitive inhibitors of the ATP-binding site and of the substrate binding site.
274
Q

List the steps of the MAP kinase cascade

A

RTK –> Grb2/SOS –> Ras –> Raf –> MEK –> MAPK

275
Q

Definition: Mitogens

A

Mitogens: signaling molecules that induce cell proliferation such as growth factors, estrogen and testosterone, Wnts

276
Q

Describe the Ras family members

A
  • All of the Ras family members are ‘small’ G proteins (170 aa) or monomeric G proteins to distinguish them from the large Gα (300 aa) proteins associated with G proteincoupled receptors.
  • Ras Is a Key Component of the MAP Kinase Cascade
  • All members function as binary switches
277
Q

How do GTPaseActivating Proteins (GAPs) affect Ras?

A

reduce Ras activation by increasing its ability to hydrolyze GTP (~100-fold). Keep about 95% of Ras inactive in the absence of a signaling molecule. Usually called RasGAPs.

278
Q

How do Guanine Exchange Factors (GEFs) affect Ras?

A

Accelerate Ras activation by promoting the exchange of GTP for the bound GDP.

Ex: SOS

279
Q

What is The Importance of Being Turned Off (Ras)?

A
  • The intrinsic GTPase activity of Ras is weak and only hydrolyzes GTP slowly
  • GTPase Activating Proteins (GAPs, Ras-GAPs) act to enhance the weak GTPase activity of monomeric G proteins and promote conversion to the inactive state
  • All/most oncogenic forms of Ras analyzed from human cancers have mutations that interfere with the ability of Ras to interact with Ras-GAP
  • These oncogenic forms of Ras are mostly in the GTP bound (active) state and continually signal the cell to promote cell division
280
Q

Describe Activation of Ras by an RTK

A

Grb2 recognizes a specific phosphorylated tyrosine on the RTK by means of an SH2 domain and recruits Sos by means of two SH3 domains. Sos stimulates guanine nucleotide exchange, which activates Ras.

281
Q

Why is The Activation of Ras Transient?

–> Experiment

A

FRET (fluorescence resonance energy transfer) analysis demonstrated that the activation of Ras is very transient. To do this, yellow fluorescent protein (YFP) was incorporated into RAS, and GTP was linked to a red fluorescent dye and injected into cells. When RAS binds GTP, the energy emitted by YFP activates the red dye and that emits fluorescent light, which can be detected and measured. The results indicate that some activated Ras can be detected by 30 sec after EGF addition, the peak of activation is about 3 min, and all Ras is inactive by 6 min.

282
Q

What does Phosphorylation of Myc, Jun, & Fos lead to (MAP Kinase cascade)?

A

Phosphorylation of Myc, Jun, & Fos leads to the transcription of genes involved in cell proliferation. Fos and Jun heterodimerize and form the AP1 transcription factor.

283
Q

True or False: GTP-bound Ras interacts directly with the ser/thrspecific protein kinase Raf (a MAP kinase kinase kinase) to activate it

A

TRUE

284
Q

What is Raf’s only catalytic substrate

A

The protein kinase Mek (a MAP kinase kinase)

–>Mek is unusual in that it contains both ser/thr protein kinase activity and tyrosine protein kinase activity (on separate structural domains)

–> Activated Mek only has one catalytic substrate family, MAP kinases, most commonly the protein kinase Erk

285
Q

True or False: Activation of MAP kinase requires phosphorylation of a threonine and a tyrosine that are separated by only a single amino acid

A

TRUE

286
Q

What are some of MAP kinases many targets?

A

Transcription factors such as Jun, Fos, and Myc

–> activates gene transcription of genes involved in cell proliferation by phosphorylating and activating these transcription activators

287
Q

True or False: Activated MAP kinase can act on target proteins in both the cytoplasm and nucleus

A

TRUE

288
Q

List Some of the Mechanisms for Attenuation & Termination of RTK Activation

A

1) Antagonists (Herceptin®)
2) Dephosphorylation (PTP = protein tyrosine phosphatases)
3) Receptor sequestration (endocytosis)
4) Receptor internalization and degradation – ubiquitination by c-Cbl
5) Receptor inhibition (ex: PKC)