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Mechanisms of Gene Regulation > Exam 2 > Flashcards

Exam 2 Flashcards

1
Q

What are the 3 RNA polymerases found in all eukaryotic nuclei?

A

RNAPI, RNAPII, RNAPIII

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2
Q

Describe RNAPI (what it is, what genes it transcribes)

A
  • Has a cleft that can encloses DNA
  • Multi-subunit enzyme
  • rRNA
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3
Q

rRNA is responsible for ______ of total cellular transcription

A

1/2

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4
Q

What transcription factor complexes are involved in the assembly of the Pol II basal transcription
complex?

A

TFIIA, TFIID, TFIIB, TFIIF, TFIIE/TFIIH

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5
Q

What are the characteristics of enhancer elements? These characteristics can help define whether a DNA element is an enhancer.

A
  • Distant regulatory sequences that can increase gene expression
  • Lack promoter activity
  • Unable to direct transcription themselves
  • Works in either orientation and from upstream or downstream
  • Frequently have tissue specific activity
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6
Q

What is a silencer? How can a silencer inhibit gene expression?

A
  • Can act on distant promoters to inhibit gene expression
  • Can inhibit gene expression at the chromatin level by recruiting factors that direct the tight packing of adjacent DNA OR by binding a protein that then directly inhibits gene expression by interacting with RNA pol and its associated factors
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7
Q

What are homeotic genes and what is their importance/how can their gene expression be regulated?

A
  • Genes that influence body patterning
  • They encode proteins that can bind DNA
  • Mutations in homeotic genes block normal development
  • Can be regulated by inactivation/improper activation of TF’s
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8
Q

What can activators interact with during transcription?

A
  • TAFs in the TFIID complex
  • Mediator complexes
  • Co-activators
  • Modulators of chromatin structure such as chromatin remodeling complexes or HATS
  • Components of TFIIB, TFIIE, TFIIH
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9
Q

How is RNA polymerase pausing overcome? What factors are involved and what is the site of action?

A
  • A second phosphorylation event
  • pTEF-b is recruited and Ser2 in carboxy terminus of RPB1 is phosphorylated
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10
Q

What functions do polyadenylation signals have? Or what role(s) do they serve? What can happen if an RNA de-adenylation enzyme removes a poly(A) tail from a transcript?

A
  • Direct internal cleavage of RNA downstream of AAUAA
  • Involved in termination
  • The de-capping complex removes the 5′ cap, leading to a degradation of the RNA.
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11
Q

There are two genes that are coregulated and in close proximity, but are on separate chromosomes. Describe a method to determine chromosome conformation.

A

Use the 3C technique
- Cross-link with formaldehyde
- Purify DNA
- Cut DNA with restriction enzyme
- Ligate DNA
- Analyze DNA to identify segments that associate in cells

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12
Q

Describe a method used to show a transcription factor is binding to a particular DNA sequence.

A

Could use either DNA mobility shift assay, DNaseI Footprinting assay or chromosome immunoprecipitation

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13
Q

RPB1 is post-translationally modified on what domain? What residues? By what enzymes? What is the role of these modifications in transcription? These modifications have multiple functions. Can you name the multiple functions?

A
  • C-terminal domain
  • 5th and 2nd serine residue
  • Enzyme: TFIIH (5th) and pTEF-b (2nd)
  • Permits initiation of transcription and releases pausing (2nd serine residue), permitting elongation
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14
Q

What groove in the DNA helix does the recognition helix bind to?

A

The major groove

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15
Q

By what mechanisms can hetero-dimerization influence gene expression?

A
  • Half sites remain similar, but the spacing between the half sites changes
  • Allows for flexibility in the type of recognition element
  • Change affinity for DNA
  • Change specificity for DNA sites (due to different spacing)
  • Lead to inactivation of transcription (think MyoD complexing with ID, which has a dimerization domain but not a DNA BD)
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16
Q

What is the importance of 5’ capping of mRNA?

A

It protects the RNA from degradation, recruits complexes involved in RNA processing, export and translation initiation, and marks cellular mRNA as “self” to avoid recognition by the innate immune system

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17
Q

Can you explain what synergistic activation is?

A

When activators work together to result in a readout much greater than what either could do alone

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18
Q

What are the function of ESEs?

A
  • ESE = exon splicing enhancers
  • Needed for proper splicing
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19
Q

What type of repeats can transcription factors bind to in DNA?

A

Direct or palindromic repeats

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20
Q

Why is it beneficial to couple processes such as transcription, RNA capping, RNA splicing, and polyadenylation?

A

Ensures correct processing of RNA

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21
Q

Can you list some functions of SR proteins?

A
  • Recruit snRNPs to RNA to be spliced
  • Help to determine which splice sites are joined together
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22
Q

What ensures that mRNA travels through nuclear pore complex 5’ end first? Explain how this is accomplished

A
  • TREX
  • CBC recruits TREX
  • TREX recruits REC to 5’ end
  • RECs promote transport to cytoplasm
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23
Q

What complexes or factors control RNA transport through the nuclear pore into the cytoplasm? What do these factors interact with?

A
  • SRs bind ESEs
  • SR dephosphorylation recruits RECs
  • RECs promote transport to cytoplasm

OR

  • H2B-Ubi promotes Ubi of CPSF protein
  • Ubi-CPSF recruits REC
  • RECs promote transport to cytoplasm

OR

  • CBC recruits TREX
  • TREX recruits REC to 5’ end
  • RECs promote transport to cytoplasm
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24
Q

What is the wobble effect of tRNA? Can you give an example of codon sequences with variation in the wobble position?

A
  • The wobble effect allows one tRNA to recognize more than one codon; allows different codons to direct incorporation of same amino acid
  • Example: U at 3rd position (wobble position) binds
    normally to A but also to G
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25
Q

What types of genes are transcribed by the three different RNA polymerases?

A
  • I: ribosomal RNA
  • II: all protein-coding genes
  • III: transfer RNA, 5S ribosomal
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26
Q

Define/describe small nuclear RNAs

A
  • 56-217 bases in size, uridine rich
  • Localized in nucleus
  • Important for splicing
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27
Q

Define/describe RNA splicing

A

A post transcriptional process by which introns and intervening sequences are removed from the RNA transcript

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28
Q

Define/describe transfer RNAs

A

A type of RNA molecule that helps decode a messenger RNA (mRNA) sequence into a protein

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29
Q

Define/describe rRNA

A

Molecule in cells that forms part of the protein-synthesizing organelle known as a ribosome and that is exported to the cytoplasm to help translate the information in messenger RNA (mRNA) into protein

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30
Q

Define/describe spliceosome

A
  • A complex structure that consists of 5 RNAs and over 100 proteins
  • Catalyzes splicing
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31
Q

Define/describe RNA polymerase

A

Enzymes that can copy the DNA into RNA together with a variety of TFs that can stimulate or inhibit polymerase activity

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32
Q

Define/describe holoenzyme

A
  • A complete, functional enzyme that is catalytically active
  • Apoenzyme + cofactors
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33
Q

Define/describe TAFs

A
  • TBP-associated factors
  • forms the TFIID complex with TBP
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34
Q

Define/describe nucleolus

A
  • Found within the cell’s nucleus
  • Contains several hundred copies of rRNA genes
  • Has proteins involved in transcription of rRNA genes by Pol I and proteins involved in the processing of 4S RNA into 28S, 18S and 5.8S rRNAs
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35
Q

Define/describe p-TEFb

A
  • A kinase that is recruited and phosphorylates RNAP II so that transcription continues and a full RNA transcript is produced
  • Required for transcriptional elongation
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36
Q

Define/describe polymerase pausing

A

A mechanism that cells use to stop and synchronize transcription

  1. Phosphorylation of Ser5 by TFIIH permits initiation of transcription
  2. Recruitment of kinase pTEF-b
  3. Phosphorylation of Ser2 permits elongation of transcription
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37
Q

Define/describe polyadenylation signal

A

AAUAA

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38
Q

Define/describe core promoter

A
  • A region of DNA close to transcriptional start site which directs transcription
  • Sequences adjacent to the transcriptional start site that control the expression of a gene
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39
Q

Define/describe upstream promoter elements

A
  • From -300 to -100
  • Upstream/before core promoter
  • May serve as targets for transcription factor binding or may interact directly with the core transcriptional machinery or both
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40
Q

Define/describe DNA mobility shift assay

A
  • Radioactively label DNA fragment
  • Mix with whole cell or nuclear extract and incubate
  • Run the mixture on a nondenaturing electrophoresis gel and observe the position of the radioactive bands by autoradiography
  • Detect protein binding to the DNA by the appearance of a shifted band
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41
Q

Define/describe DNase1 foot-printing assay

A
  • Radioactively label DNA fragment or oligonucleotide with sequence of interest at one end only
  • Mix with whole cell/nuclear extract and incubate
  • Briefly digest with DNase1, producing a series of fragments of lengths differing by one nucleotide
  • Run on a denaturing gel and detect radioactive bands by autoradiography
  • Areas with gaps indicate the presence of a protein
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42
Q

Define/describe silencers

A

A regulatory DNA sequence that can decrease the rate of transcription of a structural gene

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43
Q

Define/describe DNA binding domain

A

An independently folded protein domain that contains at least one structural motif that recognizes double- or single-stranded DNA

43
Q

Define/describe expression vector

A

Usually a plasmid or virus designed for gene expression in cells, used to introduce a gene/TF to a target cell

44
Q

Define/describe homeodomain

A
  • A DNA binding domain made up of 60 amino acids
  • contains a helix-turn-helix motif
45
Q

Define/describe recognition helix

A

A helix within the helix-turn-helix that mediates sequence specific binding and binds to the major groove

46
Q

Define/describe co-repressor

A

A protein that does not itself bind DNA but assembles on other DNA-bound gene regulatory proteins to inhibit the expression of a gene. Can mediate the effect of DNA-bound repressors

47
Q

Define/describe zinc-finger

A
  • 30 amino acid repeating sequence
  • 2-cysteine 2-histidine
  • found in many TF’s
  • zinc finger is anchored at its base with conserved cysteine and histidine residues that coordinate zinc
  • consists of 2 antiparallel beta sheets and an alpha helix
48
Q

Define/describe beta-sheets

A

Important for coordinating zinc

49
Q

Define/describe alpha-helix

A
  • Important for recognizing DNA
  • Contacts DNA major groove
50
Q

Define/describe homodimer

A

A protein composed of two polypeptide chains that are identical in the order, number, and kind of their amino acid residues

51
Q

Define/describe leucine zipper

A
  • A dimerization domain that allows DNA binding by the adjacent basic domain
  • Does not bind directly to DNA
52
Q

Define/describe helix-loop-helix

A
  • Does bind directly to DNA
  • A motif that is found in a number of transcription factors which regulate gene expression during embryonic development
  • DNA binding domain when found in homeodomains
53
Q

Define/describe MyoD

A

A TF/gene that’s ability to stimulate gene expression is inhibited by heterodimerization with the Id factor, which has a helix-turn-helix domain but no basic domain

54
Q

Define/describe domain-swapping

A

A technique with the goal of identifying activation domains
- Combine DNA-binding domain of 1 with different regions of 2
- Test on gene carrying binding site for Factor 1
- Look for gene activation

55
Q

Define/describe basal transcription complex

A
  • Considered to be the basic set of proteins needed for transcription in eukaryotes
  • Consists of RNA Pol II, TFIIA, D, B, F, E, and H
  • First TFIIA and TFIID bind to the promoter, followed by TFIIB, TFIIF/RNA Pol II and TFIIE/TFIIH
56
Q

Define/describe a mediator

A

A multiprotein complex that functions as a transcriptional coactivator in all eukaryotes

57
Q

Define/describe HIV, HI

A

Viral Tat protein
- Binds viral RNA
- Promotes Pol II initiation
- Overcomes pause
- Enhances elongation

58
Q

Define/describe a super elongation complex (SEC)

A
  • Multiprotein complex that contains pTEF-b
  • Is required for the proper induction of the HSP70 gene upon stress, and this complex is also involved in HIV proviral transcription
  • Plays a key role in stimulating transcriptional elongation of cellular as well as viral genes
59
Q

Define/describe polycistronic

A

Containing the genetic information of a number of cistrons; encodes more than one protein

60
Q

Define/describe monocistronic

A
  • A type of messenger RNA that can encode only one polypeptide per RNA molecule
  • mRNA in eukaryotes are typically monocistronic
61
Q

Define/describe Shine-Dalgarno sequence

A
  • Ribosomal binding site in bacterial messenger RNA
  • It enables initiation of protein synthesis by aligning the ribosome with the start codon
62
Q

Define/describe CPSF

A

The central component of the 3′ processing machinery for polyadenylated mRNAs: CPSF recognizes the polyadenylation signal AAUAAA, providing sequence specificity in both pre-mRNA cleavage and polyadenylation, and catalyzes pre-mRNA cleavage

63
Q

Define/describe cap-binding complex

A

Binds to the capped RNA in the nucleus, protects it from degradation and facilitates its transport to the cytoplasm

64
Q

Define/describe poly (A) polymerase

A

The enzyme that adds a chain of adenines (poly A tail) to the 3’ end of an RNA transcript, making the RNA more stable and protecting it from degradation

65
Q

Define/describe lariat

A

A lasso‐shaped structure formed from the fragment of RNA to be excised during the process of splicing out the introns in the primary transcript of eukaryotic genes

66
Q

Define/describe branch point

A

The A residue where the lariat closes itself to form a loop

67
Q

Define/describe snRNPs

A

RNA-protein complexes that combine with unmodified pre-mRNA and various other proteins to form a spliceosome, a large RNA-protein molecular complex upon which splicing of pre-mRNA occurs

68
Q

Define/describe splice site

A

The junction between the upstream exon and beginning of an intron

69
Q

Define/describe exon-splicing enhancers (ESE)

A

Facilitate exon definition by assisting in the recruitment of splicing factors to the adjacent intron

69
Q

Define/describe SR proteins

A
  • S = serine, R = arginine
  • Have 3 functions/roles: bind ESEs, recruit snRNPs and direct splice site joining
70
Q

Define/describe CTD

A
  • C terminal domain
  • largest subunit of RNA Pol II, has been demonstrated to play important roles in capping, splicing, and 3′ processing of mRNA precursors
71
Q

Define/describe exon skipping

A
  • Occurs when there is a mutation in ESE
  • Exon not bound by SR is spliced out
  • Associated with human diseases
72
Q

Define/describe RNA exporter complex (REC)

A
  • Needed for a fully processed mRNA to be transported through the nuclear pore
  • Involved in various post-transcriptional processes like capping, adenylation, and splicing
73
Q

Define/describe TREX

A

Helps RNA 5’ end pass through nuclear pore first

73
Q

Define/describe wobble effect

A

A hypothesis that describes how one amino acid can be encoded by several codons

74
Q

Define/describe initiator tRNA

A

It reads the start codon, allowing the initiating ribosome to begin translation in the correct location

75
Q

Define/describe proof reading

A

The process by which incorrect or mismatched bases are corrected

76
Q

Define/describe exosome

A

-In nucleus: a complex that degrades introns and
incorrectly spliced RNAs
- In cytoplasm: complex is involved in normal turnover functional mRNAs

77
Q

Define/describe nonsense mediated RNA decay

A
  • A second RNA degradation mechanism that exists in the cytoplasm
  • Recognition and degradation of nonfunctional mRNAs that are nonfunctional due to a premature stop codon
78
Q

Define/describe EJC

A
  • Complex that binds to each exon during splicing at a position 20-24 bases upstream of the junction between two exons and remains associated to it during export to the cytoplasm
  • Normal location of stop codons is downstream of last EJC
79
Q

What genes does RNA Pol II transcribe?

A

All protein-coding genes

80
Q

What genes does RNA Pol III transcribe?

A

Transfer RNA, 5S ribosomal

81
Q

Describe the association between DNA and RNA Pol I

A
  • RNA Pol I is not recruited to DNA by direct binding
  • Other protein factors bind DNA and recruit RNA Pol I via protein : protein
82
Q

Describe the recruitment of RNAPI

A
  1. Upstream binding factors (UBF) binds promoter
  2. SL1 is recruited via protein : protein interactions with UBF
  3. SL1 recruits RNAPI and transcription starts
83
Q

Describe RNA Pol III

A
  • Multi-subunit enzyme
  • Has a cleft that can enclose DNA
84
Q

Describe the association between DNA and RNA Pol III

A
  • Recruitment to DNA is NOT by direct binding
  • Other protein factors bind DNA and recruit RNA Pol I via protein : protein
85
Q

Describe the recruitment of RNAPIII

A
  1. TFIIIA binds promoter, then TFIIIC
  2. TFIIIC recruits TFIIIB to form a stable transcription complex
  3. TFIIIB recruits RNA Pol III via protein : protein interaction
86
Q

Describe the recruitment of RNAPII / the assembly of the basal transcription complex

A
  1. TFIIA recruits TFIID to promoter
  2. TFIID binds TATA box
  3. TFIIB recognizes TFIID/DNA
  4. TFIIB binds BREs on either side of that TATA box
  5. TFIIB recruits RNAPII / TFIIF
  6. TFIIE/TFIIH recruitment forms pre-initiation complex
  7. TFIIH kinase activity phosphorylates RNAPII C-terminal domain of RPB-1
  8. Phosphorylation permits initiation of transcription
87
Q

What is the TATA box?

A

DNA target for progressive assembly of basal pre-initiation complex of Pol II

88
Q

Describe the 3D structure of TFIID

A
  • Contains TATA binding protein (TBP)
  • Binds TATA box
  • DNA fits in groove
89
Q

What is the role of TFIIA in the assembly of the basal transcription complex?

A

Recruits TFIID to the promoter

90
Q

What is the role of TFIID in the assembly of the basal transcription complex?

A

Binds the TATA box

91
Q

What is the role of TFIIB in the assembly of the basal transcription complex?

A
  • Recognizes TFIID/DNA
  • Binds BREs on either side of TATA box
  • Recruits RNAPII / TFIIF
92
Q

What is the role of TFIIE / TFIIH in the assembly of the basal transcription complex?

A

Their recruitment forms the pre-initiation complex

93
Q

What is the role of TFIIH in the assembly of the basal transcription complex?

A

Has kinase activity, phosphorylates RNAPII C-terminal domain of RPB1

94
Q

What portion of RPB1 is phosphorylated?

A

The 5th and 2nd serine

95
Q

TFIIB is equivalent to ______ for Pol I genes

A

SL1

96
Q

RNA Pol IV and V work together to…

A

inactivate chromatin and inhibit gene expression via DNA methylation, deacetylation and/or methylation of H3 K9/K27

97
Q

Describe how TBP is a basic TF used by all polymerases

A
  • Pol I: part of SL1
  • Pol II: part of TFIID
  • Pol III: part of TFIIIB
98
Q

What are transcription factories?

A
  • Punctuate areas in nucleus
  • Genes move to these sites to be transcribed
99
Q

T / F : genes located on different chromosomes can co-localize to the same transcription factories

A

T

100
Q

What are the 3 classes of RNA Pol II distribution on genes? Which one is paused?

A
  1. Inactive gene
  2. Potentially active gene (paused)
  3. Active gene
101
Q

What are the 4 stages of transcription?

A
  1. Initiation
  2. Pausing
  3. Elongation
  4. Termination
102
Q

Define/describe co-activators

A

Proteins that do not themself bind DNA but assembles on other DNA-bound gene regulatory proteins to activate the expression of a gene. Can mediate the effect of DNA-bound activators

Mechanisms of Gene Regulation (4 decks)

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