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BIOC 4332 COPY > Exam 2 > Flashcards

Exam 2 Flashcards

1
Q

True or False: Both intron and exon sequences are transcribed into RNA.

A

TRUE: The introns are removed during processing of RNA into mRNA

–> This is called RNA splicing

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2
Q

True or False: Both replication and transcription use a DNA template to incorporate nucleotide triphosphates into a polynucleotide chain.

A

TRUE

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3
Q

True or False: Both replication and transcription use enzymes that synthesize polynucleotides by forming 3’-5’-phosphodiester bonds in the 5’ to 3’ direction. Energy derived from hydrolysis of the α-β phosphodiester bond of incoming nucleotide

A

TRUE

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4
Q

True or False: Only replication (not transcription) needs to unwind DNA

A

FALSE: Both need to unwind DNA in order to work

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5
Q

True or False: Only transcription (not replication) begins at specific sites in DNA

A

FALSE: BOTH start at specific sites

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6
Q

True or False: Both replication and transcription require the step-wise assembly of a multi-component protein complex to initiate

A

TRUE

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7
Q

What do exons contain?

A

Exons include the codons PLUS the 5’- and 3’-untranslated sequences (UTRs)

–> Therefore, introns are the spaces between exons, NOT the spaces between genes!

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8
Q

True or False: Introns and Exons are about the same size

A

FALSE: Introns are usually much longer than exons

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9
Q

When are introns removed in short transcripts?

A

Often removed from the primary RNA (pre-mRNA) after polyadenylation

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10
Q

When are introns removed in long transcripts?

A

Often removed co-transcriptionally (during transcription)

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11
Q

What processes are done BEFORE any splicing occurs in RNA?

A
  • Poly-A tailing (~200 A’s) - 3’ End

- G-capping - 5’ End

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12
Q

Define splicing

A

Removal of the intron between 2 adjacent exons

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13
Q

Define alternative splicing

A

Production of different RNAs from the same gene by splicing the RNA in different ways (ex: splicing together of non-adjacent exons

–> About 95% of human genes are spliced in this way

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14
Q

Define trans-splicing

A

Exons from two different RNAs are spliced together. This occurs in only a few eukaryotes such as trypanosomes, C. elegans

Note: This DOESN’T HAPPEN in humans

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15
Q

How does splicing increase biodiversity?

A
  • Allows for the emergence of new proteins over evolutionary time as a result of exon ‘shuffling’ during genetic recombination.
  • This concept is supported by all the ‘domains’ that proteins have in common
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16
Q

How does splicing increase the “functional density” per gene?

A

It allows one gene to make many different kinds of RNAs and thus proteins (alternative splicing). Approximately 95% of human genes are thought to be alternatively spliced

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17
Q

How does splicing get directed in long transcripts?

A

Phosphorylation of the CTD tail of RNA Pol directs RNA processing
–> Splicing occurs co-transcriptionally for most primary mRNAs

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18
Q

True or False: RNA that turns into mRNA is the only kind of RNA that gets spliced

A

FALSE: More types of RNA can be spliced

–> However, we don’t need to know about those

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19
Q

What is a transesterification?

A

A splicing event that involves 2 sequential phosphoryl-transfer reactions

–> Changes one type of phosphodiester bond to another type

–> These reactions link two exons together, while removing the intron as a lariat-like structure

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20
Q

In what situation would more than one intron be removed at a time?

A

In the case of alternative splicing, where both exons and introns might be spliced out

–> Typically, each splicing event removes only 1 intron

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21
Q

Describe how a lariat is formed and then released

A
  • A specific Adenine nucleotide in the intron (highly conserved) attacks the 5’-splice site and cuts the sugar-phosphate backbone of the RNA
  • The cut 5’ -end of the intron becomes covantly linked to the A, creating a loop
  • The free 3’-end of the 5’ exon reacts with the start of the next exon, joining the 2 exons and releasing the intron as the lariat
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22
Q

What three parts of the primary RNA transcript must the splicing machinery recognize?

A

i) The 5’ splice site (5’ of the intron to be spliced out)
ii) The 3’ splice site (3’ of the intron to be spliced out)
iii) The branch point

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23
Q

How does the splicing machinery solve the problem of short and highly variable consensus sites in the primary RNA transcript?

A

The cell has other information that helps select the splice sites

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24
Q

What 3 nucleotide “motifs” are invariant within an intron?

A

i) A GU at the 5’ end of the intron
ii) A branch site, which is always A, is found 20 – 50 NT from the 3’ splice site
iii) An AG at the 3’end of the intron

–> A pyrimidine-rich region near the 3’ end of the intron is found in most cases

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25
Q

Define: Ribonucleoprotein

A

Conains both RNAs and protein

i.e. : Ribosomes

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26
Q

True or False: The spliceosome is a large ________ particle

A

Ribonucleoprotein (RNP

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27
Q

What is snRNA?

A
  • Small nuclear RNA
  •  The RNAs in spliceosomes are specialized RNAs known as small nuclear RNAs (snRNAs)
  • These snRNAs are <200 nucleotides long
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28
Q

What are snRNAs made by?

A

Some snRNAs are made by RNA pol II and others by RNA pol III

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29
Q

What are the two specialized functions of snRNAs?

A

i) Recognize the splice sites by base pairing with the primary RNA transcript
ii) Catalyze the two transesterification reactions, i.e. snRNAs are enzymes

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30
Q

What is a snRNP?

A

They are the core units of the Spliceosome

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31
Q

True or False: All proteins in each individual snRNP are unique to that specific snRNP

A

FALSE: Some of the proteins in the snRNPs are snRNP-specific; others are components of many or all snRNP

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32
Q

Why are snRNPs called U(#)?

A

Because they are U-rich (uracil-rich)

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33
Q

What does BBP stand for?

A

Branch-point Binding Protein

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34
Q

Describe the first step of splicing

A
  • Proteins are recognizing the splice sites
  • -> U1, BBP, U2AF
  • -> Binding of the U1 and U2 snRNPs
  • -> Note: the A bulges out so its 2’-hydroxyl can do transesterification 1
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35
Q

Describe the second step of splicing

A
  • Exchange of proteins for snRNP
  • -> U2 snRNP kicking out BBF and U2AFs
  • -> Addition of U4/U6 and U5 snRNPs
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36
Q

Describe the third step of splicing

A
  • Triple SNURP comes in, U1/U4 kicked out, First transesterification reaction happens
  • -> Branch point A-OH, 3’ end of the 5’ exon
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37
Q

Describe the fourth step of splicing

A
  • 2nd transesterification reaction happens, binds the exon junction complex (splicing is complete)
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38
Q

True or False: Proteins catalyze the splicing in the spliceosome

A

FALSE: U2, U5, and U6 snRNAs actually catalyze the splicing, not proteins!

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39
Q

What is an exon junction complex? Where is it in the splicing process?

A

The spliceosome directs a set of proteins to bind to the junction of the two exons– this is the exon junction complex
–> This occurs after splicing is complete

–> The EJC marks the site of a successful splice and helps determine the subsequent fate of the mRNA

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40
Q

What are the components of the triple snRNP?

A

U4, U5, and U6

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41
Q

Why does the replacement of U1 by U6 increase the accuracy of 5’-Splice site selection?

A

Because two snRNPs have to recognize the splice site

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42
Q

True or False: ATP is necessary for splicing to occur

A

FALSE: ATP is not required for the actual splicing itself, BUT ATP IS required to get the snRNPs into place at each step. ATP is used by RNA helicases to power the RNA rearrangements.

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43
Q

What is the branch site checked by in the spliceosome?

A

The branch point is checked by BBP and then by U2

–> The binding of U2 forces the A to be unpaired

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44
Q

What is a cryptic splice site?

A

Sites in the RNA that resemble real splice sites but are not actually splice sites

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45
Q

How does co-transcriptional splicing of most pre-mRNAs increase proper splice site selection?

A
  • Longer pre-mRNAs are typically spliced as they are synthesized, meaning there is less chance for screw ups
  • Helps the cell keep track of introns and exons
  • -> This is particularly important in preventing exon skipping
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46
Q

How does exon definition increase proper splice site selection?

A

The splicing machinery recognizes when the sequence is not an exon based on sequence length. Average human exons are ~150 bases whereas average intron length is ~3500 bases. Proteins, mostly the SR proteins mark off the exons as they are transcribed.

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47
Q

About how many nucleotides long is a typical exon?

A

150 nucleotides

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48
Q

What are SR proteins?

A

Proteins that contain a serine/arginine-rich domain

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49
Q

Where do SR proteins bind?

A

They bind to exon splice enhancers

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50
Q

What is the function of SR proteins?

A

Recruit U1 and U2 snRNPs to mark the exon/intron boundaries

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51
Q

What is an hnRNP? What does it do?

A
  • Heterogeneous nuclear ribonucleoproteins

- They seem to preferentially bind to introns to help distinguish them from exons

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52
Q

True or False: Alternative splicing often occurs in a tissue-specific fashion

A

TRUE

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53
Q

True or False: All of the exons in a gene can be spliced alternatively

A

FALSE: The first and last exons in a gene CANNOT be alternatively spliced!

–> This is the only way to determine the beginning and end of the gene

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54
Q

Describe negative regulation of alternative splicing

A
  • In negative regulation (no or reduced splicing at a given site), a repressor protein binds to the pre-mRNA (to a splicing silencer sequence) and blocks splicing, often causing a cryptic splice site to be used
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55
Q

Describe positive regulation of alternative splicing

A

In positive regulation (increased splicing at a given site), splicing is inefficient unless an activator protein binds in the region (to a splicing enhancer sequence) to help

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56
Q

Describe how the human disease beta-thalassemia occurs

A

Mutated splice junctions in the beta-globin gene are often the cause of the human hereditary disease beta-thalassemia. When a mutated splice site leaves an exon without a partner, an exon is skipped, a cryptic splice site is used, or a new splice site is generated

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57
Q

How does the cell distinguish between the useful mRNA and the debris?

A
  • It is the presence of certain proteins on the mRNA and the absence of others that distinguishes a good mRNA molecule from the debris
  • -> Presence of CAP-binding complexes, exon junction complexes, poly-A-binding proteins, hnRNP proteins
  • ->  Absence of snRNP proteins
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58
Q

What is “junk” RNA that should not be exported from the nucleus (because it is just leftovers from splicing)?

A
  • Introns
  • Broken RNAs
  • Aberrantly processed pre-mRNAs
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59
Q

How is “junk” RNA (aka RNA debris) removed from the nucleus?

A

By a nuclear exosome

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60
Q

What is the nuclear exosome? What does it do?

A
  • It is a large protein complex that has proteins in its core with 3’ to 5’ exonuclease activity. The exosome thus degrades RNA debris to free nucleotides
  • The nuclear exosome also processes rRNAs to their final form
  • The cytoplasmic exosome degrades cytosolic mRNAs and thus is critical in determining their half-lives
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61
Q

How is a mature mRNA exported from the nucleus (through what structure, not the process)?

A

Mature mRNAs are exported from the nucleus via nuclear pore complexes (NPCs)

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62
Q

Describe nuclear pore complexes

A
  • NPCs are symmetrical cylindrical structures composed of many copies of about 30 proteins known as nucleoporins
  • Molecules less than ~60,000 daltons can move readily through the pores, but most mRNAs and proteins are too large
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63
Q

True or False: Mature mRNAs move through the nucleus membrane passively

A

FALSE (unless the mRNA is smaller than 60k Da) - mRNA transport to the cytoplasm requires energy

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64
Q

List all of the proteins that are bound to mature mRNA before it is exported from the nucleus (not necessarily still attached when it exports, just proteins that have bound to it while it was in the nucleus)

A

i) CBC (CAP binding complex)
ii) SR proteins (exon definition)
iii) poly A binding proteins
iv) hnRNP (heterogeneous nuclear ribonuclear proteins)
- -> Bind to newly synthesized RNA mostly in introns and compact it
v) Nuclear export receptor

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65
Q

What does the nuclear export receptor do? Where does it go when it is done doing this?

A
  • Binds to nucleoporins and thus brings the mRNA to the nuclear pore
  • Goes back to the nucleus after it’s done doing this
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66
Q

True or False: After export from the nucleus, the mature mRNA is immediately recognized by initiation factors involved in translation

A

TRUE

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67
Q

How is a gene defined?

A

Genes are defined by short sequences in the DNA that specify where RNA polymerase starts transcription (promoters) and stops transcription (terminators)

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68
Q

What is the purpose of DNA footprinting?

A

A technique for identifying the site within a region of DNA to which a specific DNA-binding protein binds

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69
Q

What is the principle behind DNA footprinting?

A

Binding of a protein to a specific site on the DNA ‘protects’ the DNA from in vitro cleavage by the endonuclease DNase I or by chemical agents

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70
Q

Why does DNA footprinting work?

A

This assay works because most DNA binding proteins have a very high affinity for their specific binding site and hence binding is stable during the time frame of the cleavage reaction

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71
Q

List the 5 steps of DNA footprinting

A
  1.   Isolate a DNA fragment of 200 - 400 bp containing the putative binding site
  2.   Label the DNA fragment at one end of one strand with radioactive or fluorescent tag
  3.   Incubate DNA fragment and protein of interest to allow binding
  4.   Treat DNA alone or protein/DNA mixture with a cleavage reagent (e.g., DNase I) so that each molecule is cleaved approximately once
  5.   Separate cleaved DNA by electrophoresis and visualize cleavage pattern by following labeled DNA strand
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72
Q

Where does the “footprint” part of DNA footprinting come from?

A

RNA polymerase gives a ‘footprint’ for where it binds to the promoter of a gene (you can’t see this region on a gel because the RNA polymerase is bound to it)

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73
Q

What is the formula for the probability of finding a specific site in the DNA?

– This tells you how long a DNA binding site has to be in order to be specific

A

(1/4)^n

where n = # of bp

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74
Q

What is a consensus sequence?

A

A consensus sequence represents the most commonly observed bases when aligning multiple sequences

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75
Q

True or False: Promoters of Prokaryotic Genes Contain Two Conserved 6 Base Pair Sequences

A

TRUE: The -10 and the -35 sequences

–> Not 100% conserved, but generally conserved

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76
Q

Define promoter strength

A

Promoter strength indicates how often transcription is initiated per unit time

  • ‘strong’ promoters have a high rate of transcriptional initiation and a good fit to the consensus
  • ‘weak’ promoters have a low rate of transcriptional initiation and a poorer fit to the consensus sequence
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77
Q

How is DNA binding affinity determined?

A

DNA binding affinity is determined by both basespecific and backbone (nonbase specific) interactions

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78
Q

True or False: “Up” mutations in the promoter of a gene decrease RNA synthesis

A

FALSE: “Up” mutations INCREASE RNA synthesis

–> “Down” mutations decrease RNA synthesis

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79
Q

How does an Up or Down mutation in a promoter molecularly change the transcription rate?

A
  • Down mutations result in poorer ‘fit’ with the consensus sequence of the promoter
  • Up mutations result in better ‘fit’ with the consensus sequence of the promoter
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80
Q

Describe the core enzyme in RNA polymerases

A

Competent for transcription elongation, but not able to recognize promoter

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81
Q

Describe the holoenzyme in RNA polymerases

A

Competent for transcription initiation; σ factor dissociates after new RNA strand reaches ≈10 nt

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82
Q

True or False: Sigma factor provides specificity to promoter recognition by RNA polymerase

A

TRUE - σ factor directly contacts the DNA at the promoter site in the -35 and -10 regions

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83
Q

True or False: Binding of RNA polymerase to the promoter can occur when sigma factor is free in the cell

A

FALSE: This binding only occurs when σ factor is present in the holoezyme complex; not when it is ‘free’ in the cell

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84
Q

What is the consensus sequence of prokaryotic promoters?

A

TTGACA (N)17 TATAAT

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85
Q

True or False: Alternative Sigma Factors in E. coli Can Direct Transcription to a Set of Genes under Unique Environmental Conditions

A

TRUE

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86
Q

True or False: Alternative sigma factors associate with same core RNA polymerase, but recognize distinct promoters

A

TRUE

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87
Q

What does σ70 recognize?

A

Most genes

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88
Q

What does σ32 recognize?

A

Genes induced by heat shock

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89
Q

What does σ28 recognize?

A

Genes for stationary phase and stress response, as well as genes involved in motility and chemotaxis

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90
Q

What does σ54 recognize?

A

Genes for nitrogen metabolism

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91
Q

What does σ24 recognize?

A

Genes for dealing with misfolded proteins in the periplasm

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92
Q

Describe negative regulation of transcription

A
  • In the absence of any regulatory factors, the gene is transcribed (on)
  • Control involves a repressor that inhibits transcription only under certain conditions
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93
Q

Describe positive regulation of transcription

A
  • In the absence of any regulatory factors, the gene is not transcribed or transcribed weakly
  • Control involves an activator that increases the ability of RNA polymerase to bind to promoter
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94
Q

True or False: Genes encoding enzymes of a specific biochemical pathway are often clustered into an operon that is transcribed from many promoters

A

FALSE: Genes encoding enzymes of a specific biochemical pathway are often clustered into an operon that is transcribed from a SINGLE promoter

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95
Q

What s a polycistronic mRNA?

A
  • Encodes multiple proteins

- Polycistronic mRNA is the transcriptional product of an operon

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96
Q

True or False: Each coding sequence has shares translational initiation and termination sites with other coding sequences

A

FALSE: Each coding sequence has its OWN translational initiation and termination site

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97
Q

True or False: Clustering of genes into an operon provides a means of coordinately controlling expression by regulating initiation from a single promoter

A

TRUE

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98
Q

How many genes does the trp operon control?

A

Five

–> For the biosynthesis of tryptophan

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99
Q

True or False: The trp repressor binds to its operator only in the presence of tryptophan

A

TRUE

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100
Q

What is the most common DNA binding domain in prokaryotic DNA binding proteins?

A

Helix-turn-helix

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101
Q

What does the binding of tryptophan to the trp repressor do?

A

It causes a minor conformational change in the repressor which allows it to bind to DNA and prevent transcription

–> Repositioning of two critical α helical segments of the trp repressor upon binding its co-repressor allow it to bind to the DNA helix

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102
Q

Is the trp repressor inducible or non-inducible?

A

It is inducable (requires a co-factor to bind it before it can bind to the trp operon)

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103
Q

What does the lac operon encode?

A

The lac operon encodes 3 genes required for utilization of lactose as an alternative energy source when glucose is absent

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104
Q

What test was done to determine that the lac repressor binds to the lac operon at a site overlapping the promoter site that blocks binding by RNA polymerase?

A

DNA footprinting

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105
Q

True or False: Mutations that define the operator site fall within the DNA footprint for lac repressor

A

TRUE

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106
Q

Define asymmetry in relation to transcription

A

Transcription is asymmetric: for any gene, only one strand of DNA is ever transcribed

–> This DNA can be either the top or bottom strand

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107
Q

Approximately what percentage of the genome gets transcribed?

A

1 - 10%

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108
Q

True or False: RNA polymerase requires a primer to be activated

A

FALSE: RNA polymerase can initiate de novo – it DOES NOT require a primer to initiate transcription

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109
Q

How many copies of the gene does trancription make?

A

2 - 1000 (varies for different genes)

–> This is different from replication, which always produces only one copy

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110
Q

What are some factors that can affect gene transcription?

A

i) Environment of the cell

ii) Cell type

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111
Q

During what phase(s) of the cell cycle does trancription occur?

A

G1 and G2

–> Replication occurs during S phase

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112
Q

What are the major modifications that occur to all eukaryotic RNA post-translationally?

A
  • 5’ capping
  • Poly-A tail (3’ end)
  • Introns spliced out
  • Transport out of the nucleus and into the cytoplasm
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113
Q

Describe “step 0” of transcription

A

RNA polymerase (RP) binds weakly to DNA and scans for a promoter

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114
Q

Describe “step 1” of transcription

A

Sigma factor aids the RNA polymerase promoter recognition by binding directly to the promoter sequence

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115
Q

Describe “step 2” of transcription

A

RNA polymerase and sigma factor unwind 10 - 12 bp of DNA

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116
Q

Describe “step 3” of transcription

A

Initiation involves formation of phosphodiester bonds between first 2 nucleotide pairs. “Scrunching” causes stress and necessitates starting over a few times

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117
Q

Describe “step 4” of transcription

A

After the RNA polymerase has coupled about 10 nucleotides a few times, it breaks free of the promoter sequence and weakens the interaction with sigma factor

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118
Q

Describe “step 5” of transcription

A

Sigma factor dissociates after RNA polymerase has escaped from promoter

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119
Q

Describe “step 6” of transcription

A

During elongation phase, RNA polymerase synthesizes RNA in a highly processive fashion (about 50 nucleotides per second)

  • -> However, RP has a much higher error rate than DNA polymerase (~1/104 nucleotides)
  • -> DNA in front of RNA polymerase becomes positively supercoiled while trailing DNA becomes negatively supercoiled
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120
Q

What do topoisomerases do in transcription?

A

They relieve supercoiling

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121
Q

True or False: RNA polymerase must completely synthesize a new RNA strand for a gene before another RNA polymerase can begin to synthesize (prokaryotes)

A

FALSE: Once RNA polymerase has cleared the promoter, another RNA polymerase complex can bind the promoter and initiate transcription, etc.

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122
Q

Describe “step 7” of transcription

A

RNA polymerase reaches the terminator sequence, which consists of two parts:

i) A GC-rich inverted repeat
ii) 6 - 8 consecutive adenines

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123
Q

What is a GC-rich inverted repeat?

A

It is part of the transcriptional terminator sequence, which forms a stable hairpin formation that causes the RNA polymerase to pause

124
Q

What do the 6 - 8 consecutive adenines do in the transcriptional terminator?

A

When RNA polymerase is paused because of the GC-rich hairpin loop, it is situated on top of this consecutive adenine sequence.

Dissociation of the RNA polymerase then occurs because of the weak rU-dA complex in the active site of RNA polymerase

125
Q

What is the eighth and final step of transcription?

A

After release of the RNA polymerase core enzyme, it is free to re-associate with Sigma factor and initiate another round of transcription.

126
Q

How many different non-covalent contacts will form in a typical DNA-protein interaction?

A

20-30

–> The combination of all of these ends up in multivalency (a high-affinity overall interaction)

127
Q

What is a consequence of the multivalency of the interactions between proteins and the back bone of DNA?

A

All sequence-specific DNA binding proteins also have a general affinity for any DNA sequence

128
Q

True or False: 5’ Capping and Poly-A tailing occur in both prokaryotes and eukaryotes

A

FALSE: These only occur in EUKARYOTES

–> Because RNA needs to be transported out of the nucleus in eukaryotes– this is not an issue for prokaryotes

129
Q

True or False: RNA polymerase terminates more precisely in eukaryotes than in prokaryotes

A

FALSE: It is more precise in prokayotes– eukaryotes under a bunch of post-translational modification, so they don’t necessarily need to be super precise

130
Q

Describe the process of 5’ Capping

A

i) First, a phosphatase removes the γ-phosphate from the most 5’ nucleotide of the pre-mRNA.
ii) Next, a guanylate transferase adds GMP in an unusual linkage.
iii) Finally, a methyl transferase adds one methyl group to the guanosine

131
Q

At what time (how many nucleotides into transcription) does 5’ Capping occur?

A

The 5’ cap is added when the nascent pre-mRNA is about 25 nucleotides long

132
Q

Describe poly-A tailing (molecular mechanisms)

A

i) Cleavage and Polyadenylation Specificity Factor (CPSF) and Cleavage Stimulatory Factor (CStF) first bind to the phosphorylated CTD of RNAPII
ii) CPSF then binds the AAUAAA element.
iii) CStF binds the G/U rich element.
iv) Additional factors bind and cleave the RNA
v) Next poly A polymerase (PAP) binds to the 3’ end and adds ~200 adenosines one at a time (no template required!)
vi) Poly-A-binding proteins then bind to poly-A tail

133
Q

What does the Poly-A tail do for the mRNA?

A

Poly-A-binding proteins play a role in mRNA stability, quality control and translation initiation

134
Q

At what point in the degradation of the Poly-A tail (by deadenylase) does the mRNA itself begin to degrade?

A

When there are only about 25 adenines left in the Poly-A tail

–> Note: This is the number for humans, not necessarily for all eukaryotes

135
Q

What are the most important roles that the 5’ cap and the poly-A tail have?

A
  • The 5’ cap protects the mRNA from degradation by 5’-3’ nucleases.
  • The poly(A) tail protects the 3’ end from 3’-5’ nucleases
  • Both the 5’ cap and poly-A tail are required for translation initiation
136
Q

What enzyme degrades mRNA?

A

Deadenylase

137
Q

How many enzymes make up RNA polymerase (prokaryotes)?

A

One

138
Q

How many enzymes make up RNA polymerase (eukaryotes)?

A

Three:
-pol I: rRNA
pol II: mRNA
pol III: tRNA, etc

139
Q

What is the promoter for transcription (prokaryotes)?

A

-35 box and -10 box

140
Q

What is the promoter for transcription (eukaryotes)?

A

TATA box (pol II) and more variable set of additional DNA elements

141
Q

What proteins are required for initiation of transcription (prokaryotes)?

A

Sigma factor

142
Q

What proteins are required for initiation of transcription (eukaryotes)?

A

Many GTFs and specific transcription factors and co-factors

143
Q

What kinds of RNA processing occurs for prokaryotes?

A

None

144
Q

What kinds of RNA processing occurs for eukaryotes?

A

Poly-A tail, 5’ G-capping, Splicing

145
Q

Define catabolite repression

A

The repression of a certain gene in the presence of a certain catabolite

I.e. the lac operon is catabolically repressed in the presence of glucose

146
Q

What controls catabolite repression for the lac operon in E. coli?

A

Catabolite repression is controlled by the Catabolite Activator Protein (CAP) and cAMP, which binds to CAP

147
Q

True or False: The synthesis of cAMP by adenylate cyclase is (increased/decreased) in bacteria when glucose is depleted from its environment

A

The synthesis of cAMP by adenylate cyclase is INCREASED in bacteria when glucose is depleted from its environment

148
Q

What is the purpose of cAMP in relation to the lac operon?

A

cAMP is an intracellular signal that glucose supplies have been exhausted and the cell better start using alternative carbon sources

149
Q

Where does CAP bind to the lac operon?

A

CAP binds to the lac operon at an upstream site immediately adjacent to the promoter

150
Q

How does cAMP-CAP binding to the lac operon affect DNA structure?

A

cAMP-CAP binding bends DNA and helps to recruit RNA polymerase

151
Q

True or false: The lac operon is both positively and negatively controlled

A

TRUE

152
Q

When glucose is _____, cAMP levels RISE and bind/ activate binding of _______ protein at promoter

A

When glucose is LOW, cAMP levels rise and bind/ activate binding of CAP protein at promoter

153
Q

When lactose is LOW, the ______ has no lactose bound and BINDS to the operator to (initiate/block) polymerase progression

A

When lactose is low, the REPRESSOR has no lactose bound and BINDS to the operator to BLOCK polymerase progression

154
Q

When lactose is HIGH, lactose binds the ________ and causes an allosteric change, making it (able/unable) to bind the operator

A

When lactose is high, lactose binds the REPRESSOR and causes an allosteric change, making it UNABLE to bind the operator

155
Q

What are the two critical proteins that control expression of the lac gene?

A

Lac repressor and CAP

156
Q

How does the lac repressor function?

A

In the absence of lactose, it is bound to the operator. When lactose is present, lactose binds to the repressor and causes it to release from the operator, allowing the lac gene to be transcribed

157
Q

How does CAP function?

A

Low glucose allows cAMP to accumulate. cAMP activates CAP, CAP binds to the RNA polymerase and allows it to bind

158
Q

What is the term for the type of multi-gene-containing mRNA that is produced when the lac operon is activated?

A

Polycistronic

159
Q

Describe the experiment that was done to prove that all DNA is present in every cell, even though different cell types exist

A
  • Took an unfertilized egg (from a frog) and removed genetic content
  • Put in a nucleus from a skin cell into the egg
  • The egg grew into a regular frog, not into just one skin cell
160
Q

Describe regulation of gene expression in eukaryotic organisms (5 things)

A
  • Eukaryotic cells generally have more genes and a lot more DNA than prokaryotic cells
  • DNA in eukaryotic cells are packaged into complex chromosomal structures that limit accessibility of the transcriptional machinery
  • Complex organisms possess multiple cell types with specialized function determined by differences in gene expression
  • Development and differentiation are largely regulated by temporal and spatial changes in gene expression
  • Many cell signaling pathways affect cell function and physiology by altering patterns of gene expression
161
Q

Which eukaryotic RNA polymerase is most important for gene expression?

A

RNA polymerase II

–> Transcribed mRNA and some snRNAs

162
Q

Which eukaryotic RNA polymerase transcribes pre-RNA?

A

RNA polymerase I

163
Q

Which eukaryotic RNA polymerase transcribes tRNA?

A

RNA polymerase III

164
Q

How many subunits (approximately) make up each RNA polymerase?

A

12-16 subunits

–> Some are in all three types, some are unique for each

165
Q

Why does RNA polymerase II face the most complex regulatory job of the three polymerases?

A

It needs to recognize over 25,000 different promoters and transcribe these genes at highly different rates depending on the needs of the cell

166
Q

What is the main difference between eukaryotic RNA polymerase II and prokaryotic RNA polymerase?

A

Regulatory features of RNA polymerases are what differs between them

167
Q

What are the four conserved sequences at the promoter in eukaryotic cells? Which TFII(x) recognizes these sequences?

A

i) BRE - TFIIB
ii) TATA box - TFIID
iii) INR - TFIID
iv) DPE - TFIID

168
Q

What does BRE stand for?

A

B recognition element

–> Where TFIIB binds

169
Q

What does INR stand for?

A

Initiator Motif

170
Q

What does DPE stand for?

A

Downstream promoter element

171
Q

True or False: Eukaryotic RNA polymerases can recognize the promoter and begin initiation of transcription without the assistance of other components

A

FALSE: Eukaryotic RNA polymerases require additional components for recognition of their promoter and initiation

172
Q

What is TFIID’s role in transcription?

A
  • Recognizes TATA box
  • Recognizes other DNA sequences near the transcription start point
  • Regulates DNA binding by TBP
173
Q

What is TFIIB’s role in transcription?

A
  • Recognizes BRE element in promoters

- Accurately positions RNA polymerase at the start site of transcription

174
Q

What is TFIIF’s role in transcription?

A
  • Stabilizes RNA polymerase interaction with TFB and TFIIB

- Helps attract TFIIE and TFIIH

175
Q

What is TFIIE’s role in transcription?

A

-Attracts and regulates TFIIH

176
Q

What is TFIIH’s role in transcription?

A
  • Unwind DNA to expose template strand at start point of transcription
  • Hydrolyzes ATP and changes conformation
  • Phosphorylates Ser5 residue of the RNA polymerase CTD
  • Releases RNA polymerase from the promoter
177
Q

What kinds of activity does TFIIH have?

A

Helicase and kinase

178
Q

What does CTD stand for?

A

C-terminal domain

179
Q

What does TFII(x) stand for?

A

Transcription factor two (x)

180
Q

Where is the TATA box found?

A

The Conserved TATA Box is Found ~30 Bases Upstream from Many RP II Start Sites

181
Q

True or False: Genes without a TATA box are unable to be transcribed

A

FALSE: Genes that lack TATA box contain other, less well conserved, consensus sequences near the transcription start point

182
Q

What transcription factor is the first to bind? What does it do when it binds?

A

TFIID

–> Nucleates formation of the initiation complex after binding

183
Q

What does TBP stand for? Which transcription factor is it a part of?

A

TATA Binding Protein

–> Part of TFIID

184
Q

What does TAF stand for?

A

TBP-associated factors

185
Q

How does TBF bind to the DNA?

A

TBP binds to the TATA box by itself recognizing the MINOR groove of DNA helix and causing a bend in the DNA

186
Q

What do the TAF subunits do during binding to the DNA?

A

TAF subunits interact with other promoter elements (e.g., DPE) to specify sites of transcriptional initiation

187
Q

Why is it helpful for TFIID to create a sharp bend in the DNA when it binds?

A

Binding of TFIID (TBP) to TATA box causes a sharp bend in DNA thought to serve as a physical marker for an active promoter in very large eukaryotic genomes

188
Q

True or False: General transcription factors act in a sequential manner to assemble the initiation complex on the promoter

A

TRUE

189
Q

Which transcription factor binds after TFIID?

A

TFIIA binds next

–> Stabilizes TFIID binding to DNA

190
Q

How does TFIIB know where to bind the DNA?

A

TFIIB recognizes complex between TFIID and DNA and binds to DNA that has been ‘bent’ by TBP

191
Q

When does TFIIF bind during the transcription sequence? What does it do after it binds?

A

TFIIF binds after TFIIB

–> Escorts RNA polymerase II to the transcription start site

192
Q

When does TFIIE bind during the transcription sequence? What does it do at this time?

A

TFIIE binds after RNA Polymerase II and acts to bring TFIIH into the initiation complex

193
Q

When does TFIIH bind during the transcription sequence? What does it do at this time?

A

TFIIH binds last

  • 2 of it’s subunits have a helicase activity (open the DNA for RNA polymerase II to transscribe)
  • 1 of its subunits has kinase activity (phosphorylates the Ser5 residue)
194
Q

True or False: The C-terminal domain is present in all RNA polymerases

A

FALSE: The CTD is found exclusively on RPII; not RPI or RPIII or any prokaryotic RP

195
Q

Where is the C-terminal domain (CTD) subject to be phosphorylated?

A

It’s subject to phosphorylation at several of its Serine residues

196
Q

What is the repeating heptapeptide on the C-terminal domain?

A

(YSPTSPS)n where n=27-52

–> This is located on the largest subunit of RNA polymerase II

197
Q

What is the point of having multiple potential CTD phosphorylation sites? Why aren’t they all always phosphorylated?

A

Proteins can recognize different patterns of phosphorylation

–> Recruitment of different proteins based on which Serines are phosphorylated

198
Q

How can DNA supercoiling be helpful in transcription?

A

The tension generated by supercoiling may facilitate unwrapping of DNA in nucleosomes, which provides access to the DNA and helps to relieve supercoil tension

199
Q

True or False: In cells, RPII is found as a large multiprotein complex containing the GTFs (general transcription factors) and many additional polypeptides

A

TRUE

200
Q

True or False: Over 100 different subunits must assemble to initiate transcription of a gene in eukaryotes

A

TRUE

201
Q

How have transcription control regions of eukaryotic genes been defined (experiment)?

A

By reporter gene assays in combination with in vitro mutagenesis

202
Q

What can a DNA microarray be used for?

A

DNA microarrays provide a means of rapidly comparing patterns of gene expression between two cell types or a single cell type under different conditions (+/- growth factor)

203
Q

True or False: Even though eukaryotic genes are not organized into operons like prokaryotic genes, there are mechanisms to coordinate the regulation of genes in pathways

A

TRUE

204
Q

For any eukaryotic gene, the rate of transcriptional initiation will be determined by (4 things):

A

i) The set of promoter proximal and enhancer elements that regulate the gene
ii) The concentration of corresponding transcription factors in a specific cell
iii) The affinity of each transcriptional activator for its DNA regulatory site
iv) Cellular regulation of transcription factor activity

205
Q

Describe the phosphorylation of the CTD tail during each stage of transcription

A

i) Initiation: No phosphoryation; Recruited to promoter by general transcription factors
ii) Promoter Clearance/Recruitment of capping factors: Phosphorylation is occuring by CTD kinases (including but not limited to TFIIH)
iii) Elongation: Phosphorylation along the tail that is recruiting the different splicing, PolyA, etc factors; Not BEING phosphorylated at this point, but the phosphorylation sites are what is recruiting the proteins
iv) Termination: CTD Phosphatases must come and remove all the phosphates from the tail, so that RNA polymerase II can bind to a new promoter and begin the journey again

206
Q

How does a eukaryotic cell deal with the superhelical tension in its genomic DNA resulting from the activity of RNA polymerases?

A

DNA topoisomerases rapidly remove the superhelical tension caused by transcription.

207
Q

True or False: The acetylation of lysines on the histone tails can be performed on methylated lysines only after they are first demethylated

A

TRUE

208
Q

True or False: The acetylation of lysines on the histone tails loosens the chromatin structure because it adds positive charges to the histone

A

FALSE

209
Q

True or False: The acetylation of lysines on the histone tails recruits the heterochromatin protein HP1, resulting in the establishment of heterochromatin

A

FALSE

210
Q

True or False: The acetylation of lysines on the histone tails is sufficient for the formation of an open chromatin structure

A

FALSE

211
Q

True or False: The acetylation of lysines on the histone tails is a covalent modification and is thus irreversible

A

FALSE

212
Q

True or False: DNA gyrase is present only in prokaryotes

A

TRUE

213
Q

Are histone proteins acidic or basic? Why does this matter?

A

Histone proteins are basic.

This is important because their basic sidechains help neutralize the negative charges on the phosphate backbone of DNA

214
Q

True or False: The chromatin remodeling complexes can slide nucleosomes on DNA

A

TRUE

215
Q

True or False: Chromatin remodeling complexes have ATPase activity

A

TRUE

216
Q

True or False: The chromatin remodeling complexes interact with histone chaperones

A

TRUE

217
Q

True or False: The chromatin remodeling complexes can remove or exchange core histone subunits

A

TRUE

218
Q

True or False: To ensure the fidelity of splicing, the spliceosome hydrolyzes ATP to undergo complex rearrangements

A

TRUE

219
Q

True or False: To ensure the fidelity of splicing, the spliceosome examines the splicing signals on the pre-mRNA several times.

A

TRUE

220
Q

True or False: To ensure the fidelity of splicing, the spliceosome assembles on the pre-mRNA co-transcriptionally

A

TRUE

221
Q

True or False: To ensure the fidelity of splicing, the spliceosome takes advantage of “exon definition.”

A

TRUE

222
Q

After the first and before the second chemical step of RNA splicing, the intron of the pre-mRNA is covalently connected to which end of the exon? What is the internal shape?

A

After the first and before the second chemical step of RNA splicing, the intron of the pre-mRNA is still covalently connected to the 3’ exon and has an internal branch in the shape of a lariat.

223
Q

The presence of which molecule(s) on an mRNA is a signal that the mRNA is still NOT ready for nuclear export?

A

snRNPs used in splicing

224
Q

The presence of which molecule(s) on an mRNA is a signal that the mRNA IS ready for nuclear export?

A
  • Exon junction complex
  • Cap-binding complex
  • SR proteins
  • poly-A-binding proteins
225
Q

What is needed for effective initiation of transcription by RNA Polymerase II?

A

Effective initiation by RP II requires multiple regulatory DNA sequences (elements) located near the promoter and at much greater distances from the initiation site

226
Q

True or False: Regulation of activators/coactivators, repressors/co-repressors is often allosteric

A

TRUE

227
Q

Define allostery as it relates to regulation of transcription

A

Structural changes due to binding of a ligand molecule or other protein

228
Q

What is a basal promoter?

A

A promoter that allows a low transcription to occur

–> Allows binding of transcriptional elements even with the main promoter is blocked

229
Q

What is a promoter proximal element?

A

Short (about 10 nt) sequences residing -200 to -40 upstream of the basal promoter that greatly increase initiation of transcription in many eukaryotic genes

–> Called promoter proximal elements (PPEs) to distinguish them from the basal promoter

230
Q

True or False: Enhancers only function when they are near to the basal promoter in order to maintain specificity of the enhancer

A

FALSE: Enhancers can function at great distances (up to 50 kb) from the basal promoter

–> Enhancers function at great distances because DNA between the enhancer and the promoter site can loop (DNA looping)

231
Q

True or False: Enhancers are only found upstream of the basal promoter that they influence

A

FALSE: Enhancers are found either upstream or downstream of the basal promoter that they influence in different genes

232
Q

True or False: In transfection assays, enhancers can function in either orientation relative to the direction of transcription

A

TRUE

233
Q

True or False: Enhancers can (usually) function when artificially linked to a promoter from a different gene

A

TRUE

234
Q

True or False: The promoter proximal region and enhancers BOTH contain short DNA regulatory elements that bind to specific transcription factors

A

TRUE

235
Q

How is the promoter region defined?

A

The promoter proximal region is defined SOLELY by its close proximity to the basal promoter

236
Q

How do enhancers differ from the promoter promixal region?

A

They only differ in their location relative to the basal promoter

237
Q

Where can enhancers be located in relation to the basal promoter?

A

Enhancers can be located anywhere -50 to +50 kb upstream or downstream of the basal promoter but not in the promoter proximal region

238
Q

True or False: Enhancers can be located in introns

A

TRUE

239
Q

What are the key components that allow eukaryotic cells to regulate initiation of transcription?

A

Activators and repressors

240
Q

What test would you run to determine the DNA binding activity of a transcription factor?

A

Electrophoretic mobility shift assay (EMSA)

241
Q

Describe an electrophoretic mobility shift assay (EMSA)

A

i) Isolate a piece of DNA containing regulatory elements for your favorite gene and label with 32P
ii) Incubate labeled DNA with nuclear extract containing transcription factors
iii) Labeled DNA fragment migrates as a single band on non-denaturing gel
iv) Subpopulations of labeled DNA will ‘shift’ to slower migrating bands (ie., greater molecular weight) due to binding of transcription factors

242
Q

Given that 6% of mRNAs encode transcription factors and their cellular abundance is very low, how can transcription factors that recognize a specific DNA sequence be found?

A

– Enrichment based on protein properties (e.g. ion exchange chromatography)
– Enrichment based on affinity to the DNA

243
Q

How can transcription factors in a nuclear extract be resolved?

A

Transcription factors in nuclear extract can first be resolved by Ion Exchange Chromatography

–> Then, individual fractions assayed for DNA binding using EMSA

244
Q

How can we assess the functional activity of specific transcription factors: i.e. their ability to alter rates of transcriptional initiation?

A

Co-transfection assays (a type of gene reporter assay)

245
Q

True or False: DNA binding regions of transcriptional activators and repressors are distinct structural domains and therefore cannot function when separated from the remainder of the protein

A

FALSE: Because of their distinct structual domains, DNA binding regions CAN function when separated from the remainder of the protein

246
Q

True or False: DNA binding domains are always directly involved in transcriptional activation

A

FALSE: DNA binding domains are (usually) NOT directly involved in transcriptional activation

247
Q

What is the main role of the DNA binding domain?

A

The main role of the DNA binding domain is to localize the appropriate specific transcription factor to the correct DNA sequence within the gene that it influences

248
Q

Describe the size and structure of the homeodomain of the DNA binding domain

A

60 amino acid, 3 helix structure with helix-turn-helix motif

249
Q

True or False: Homeodomains with very low sequence identity are structurally not very well conserved

A

FALSE: Homeodomains with very low sequence identity ARE structurally conserved

250
Q

What kind of symmetry do dimers bound to inverted repeats display?

A

Dimers bound to inverted repeats display rotational symmetry, NOT mirror symmetry!

251
Q

Describe the Zinc finger DNA binding domain

A

Zinc finger proteins usually form dimers that bind to inverted repeats

252
Q

Describe the Leucine zipper DNA binding domain

A

Leucine zippers, like zinc fingers, usually form dimer

253
Q

Describe the Helix-loop-helix DNA binding domain

A

Helic-loop-helix ALSO binds as a dimer (just like zinc fingers and leucine zippers)

254
Q

Can we predict which amino acid(s) hydrogen bond with a given base pair given all we know about the structures of DNA binding domains and their interactions with DNA?

A
  • No, although some amino acid-base Interactions occur with much greater frequency.
  • -> Arginine is one such example because it can unambiguously hydrogen bond with guanine.
255
Q

True or False: The ability to form heterodimers increases combinatorial regulation of genes

A

TRUE: This is why so many transcription factors bind DNA as dimers

–> transcription factors can be activators or repressors

256
Q

Describe how some transcription factors can function as activators

A
  • Activators consist of a DNA binding domain and another domain that binds to other proteins required to effect activation
  • Activators act via binding to either the promoter proximal or enhancer regions of genes
  • -> Activators can aid initiation of transcription via multiple mechanisms
257
Q

In what four ways can an activator act?

A

i) Promoting binding of additional regulators
i) Recruiting RNA polymerase to promoter
iii) Releases RNA polymerase to begin transcription
iv) Releases RNA polymerase from pause

258
Q

What is a transcriptional coactivator?

A

Coactivators are non-DNA binding components that serve to bridge between activators and the basal transcriptional machinery

  • -> Most coactivators can interact with multiple activators and may serve to integrate cell signals
  • -> Coactivators act either by directly recruiting GTFs to the initiation site or by modifying chromatin structure
  • -> Similarly, many repressors function by recruiting corepressors to specific promoters
259
Q

What can the coactivator CBP do?

A

The Coactivator CBP Can Integrate Inputs from Multiple Transcriptional Activators

–> Caution: Don’t confuse CREB/CPB in eukaryotes with cAMP-CAP in prokaryotes!

260
Q

Name the proteins involved in eukaryotic transcription initiation in the order in which they assemble at the promoter

A

1) TFIID
2) TFIIA
3) TFIIB
4) TFIIF
5) RNA Polymerase II
6) TFIIE
7) TFIIH

261
Q

Which transcription factor acts as a “loader” to escort the last GTF to assemble at the promoter before promoter clearance and elongation

A

TFIIE

262
Q

Which transcription factor binds to the TATA box via its TBP subunit?

A

TFIID

263
Q

Which transcription factor stabilizes the complex between the first GTF to bind at the promoter, and the DNA?

A

TFIIA

264
Q

Which transcription factor acts as a “loader” to escort RNA Pol II to the promoter?

A

TFIIF

265
Q

Which transcription factor recognizes the bent DNA and binds to the B recognition element?

A

TFIIB

266
Q

Which transcription factor is responsible for performing the templated enzymatic reaction to elongate RNA?

A

RNA polymerase II

267
Q

Which transcription factor is the last GTF to assemble at the promoter, and has helicase and kinase activity?

A

TFIIH

268
Q

How can a transcription factor function as a repressor?

A

By interfering with formation of the initiation complex

269
Q

Describe the function of a transcription factor as it is acting as a repressor

A
  • Like transcriptional activators, repressors consist of a DNA binding domain and another domain that effects repression
  • Repressors also act via binding to either the promoter proximal or enhancer regions of genes
  • Rather, eukaryotic repressors can inhibit initiation of transcription via multiple mechanisms (in different flashcards)
270
Q

True or False: Both eukaryotic and prokaryotic repressors sterically block the binding of the initiation complex to the basal promoter

A

FALSE: Unlike prokaryotic repressors, eukaryotic repressors DO NOT sterically block binding of the initiation complex to the basal promoter

271
Q

In what three ways can a transcription factor act as a repressor (just the names, not the functions)?

A

1) Competitive DNA binding
2) Masking the activation surface
3) Direct interaction with the general transcription factors

272
Q

Describe competitive DNA binding

A
  • A form of transcription factor repression
  • The binding site on the DNA overlaps for the activator and repressor
  • -> If the repressor binds first, then the activator can’t bind properly to the DNA and won’t function
273
Q

Describe masking the activation surface (transcriptional repression)

A
  • A form of transcription factor repression
  • The repressor will bind to the activator’s activation surface and therefore block transcription factors from binding to it
274
Q

Are promoter proximal elements made of nucleotides or proteins?

A

Nucleotides

275
Q

Describe direct interaction with the general transcription factors (transcriptional repression)

A
  • A type of transcription factor repression

- The repressor binds to a transcription factor and prevents it from carrying out its function

276
Q

Are enhancers made of nucleotides or proteins?

A

Nucleotides

277
Q

True or False: The mechanisms used to regulate the activity of a transcription factor are the same as those used to regulate enzyme activity

A

TRUE

–> This regulation of transcription factor activity is critical for their role in carrying out complex developmental programs or responding to changes in the environment

278
Q

What serves as the interface between cell signaling pathways and control of gene expression?

A

Transcription factors

279
Q

What can ChIP-Seq tell you?

A

Which sequences on the DNA are bound by certain proteins – used for discovery

–> Need to know only what protein you’re using, not what sequences you’re looking for

280
Q

What do you need for a cotransfection assay?

A

Two plasmids: Expression plasmid and a reporter plasmid

281
Q

Define epigenetics as it relates to biochemistry

A
  • The chemical modifications to DNA and its histones that regulate gene transcription that do not involve changing the nucleotide sequence.
  • -> These modifications are called ‘marks’.
282
Q

Define epigenetic inheritance as it relates to biochemistry

A
  • The heritable and reversible modifications that affect gene expression (transcription) and genome stability without changing the nucleotide sequence.
  • -> This is the ‘memory’ of that cell or organism’s experiences.
  • -> The memory is typically established and erased (reversed) by enzymes.
283
Q

True or False: Epigenetic events are much less frequent than genetic events (nucleotide changes).

A

FALSE: Epigenetic events are much MORE frequent than genetic events (nucleotide changes).

284
Q

Describe DNA packaging from the smallest unit to the largest unit (not too in-depth)

A

1) “Beads on a string” form of heterochromatin
2) 30-nm chromatin fiber of packaged nucleosomes
3) Extended form of chromatin
4) Condensed form of chromatin
5) Chromosome

285
Q

How can transcriptional activators recognize their binding sites even when these sites are packaged into nucleosomes?

A

These activators can function by displacing or phasing nucleosomes to provide access to TFIID (or other transcription factors)

–> Enhancer regions are nucleosome-free

286
Q

How do transcriptional activators deal with nucleosomes?

A

–> Once bound to their DNA regulatory elements, some activators can rearrange nucleosomes to provide access to other proteins

–> More commonly, activators act by recruiting other factors (coactivators, such as chromatin remodeling complexes) that modify the nucleosomal structure or organization

287
Q

What will trimethylation on lysine 9 on histone H3 do to the gene?

A

Heterochromatin formation, gene silencing

288
Q

What will the combination of trimethylation of lysine 4 and acetylation on lysine 9 of histone H3 do to the gene?

A

Gene expression

289
Q

What will the combination of phosphorylation on serine 10 and acetylation on lysine 14 on Histone H3 do to the gene?

A

Gene expression

290
Q

What will trimethylation of lysine 27 on histone H3 do to the gene?

A

Silencing of Hox gene, X chromosome inactivation

291
Q

What do coded histone tails require to recognize modification?

A

Coded histone tails require CODE READERS to recognize the modifications

292
Q

What is a chromodomain?

A

The protein domain that binds methylated lysine

–> A code reading protein

293
Q

What is a bromodomain?

A

The protein domain that binds acetylated lysine

–> A code reading protein

294
Q

True or False: Acetylation of amino-terminal ‘tails’ of four core histones is highly conserved in the cell

A

FALSE: Acetylation of amino-terminal tails of four core histones is highly DYNAMIC in the cell

295
Q

How does acetylation affect the interaction between linker DNA and adjacent nucleosome particles?

A

Acetylation neutralizes a positive charge on lysine and this may reduce interaction between linker DNA and adjacent nucleosome particle

296
Q

True or False: Nucleosomes associated with actively transcribed genes contain lower levels of acetylated histones than nucleosomes associated with inactive genes

A

FALSE: Nucleosomes associated with actively transcribed genes contain HIGHER levels of acetylated histones than nucleosomes associated with inactive genes

297
Q

How are lysines on histone tails acetylated and deacetylated?

A

Lysines on histone tails are reversibly acetylated by acetyl transferases and deacetylases

298
Q

How does recruiting coactivators to promoter regions affect chromatin accessibility?

A

Hence, by recruiting coactivators to specific promoter regions, activators can increase acetylation of histone tails and alter chromatin accessibility for other transcriptional factors

299
Q

True or False: Some transcriptional repressors function through recruitment of histone acetylases

A

FALSE: Some transcriptional repressors function through recruitment of histone DEacetylases

300
Q

True or False: Transcriptional repressors function by recruiting histone methyl transferases

A

TRUE

301
Q

How do chromatin remodeling complexes displace or remodel nucleosomes?

A

Chromatin remodeling complexes contain many subunits and use energy of ATP hydrolysis to displace or remodel nucleosomes

302
Q

True or False: Chromatin remodeling complexes and coactivators function sequentially during activation of gene transcription

A

TRUE

303
Q

With so many factors influencing transcriptional initiation, how can we identify the regions of chromatin that are actively transcribed?

A
  • Microscopically, chromatin can be divided into:
  • –  Euchromatin: more lightly staining; in a more open, accessible conformation
  • – Heterochromatin: more densely staining; in a more compacted, inaccessible conformation
  •   Actively transcribed genes are typically found in euchromatin
  •   Centromeres, telomeres and highly repetitive DNA are typically found in heterochromatin
  •   Some regions of chromatin are euchromatic in one cell type, but heterochromatic on others
304
Q

By what three methods can the PCR products obtained by ChIP can be analyzed by?

A

i) qPCR (if targets are known)
ii) Microarray (known as “ChIP-on-chip” or “ChIPchip”)
iii) next generation sequencing methodology (ChIP-Seq)

305
Q

Describe Cytosine methylation

A
  • In vertebrates, a small % of cytosine residues are methylated at the 5-position
    -  Methylation does not alter the base pairing properties of C
    -  Methylated C residues are always found as part of dinucleotide (5’)CG with the same modification occurring on both strands (“CG islands”)
    -  This provides a means of passing along a pattern of
    methylation stably during cell division
    –> DNA methylation is thought to spread in “CG islands” to silence gene transcription
306
Q

Describe heritability as it relates to thic class

A

Maintenance methylases recognize hemi-methylated DNA sites and add an additional methyl group on the opposite strand

307
Q

How can histone modifications be heritable?

A

Histone modifications can be “heritable” if parental histones are used to condense newly replicated DNA

BIOC 4332 COPY (5 decks)

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