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Eukaryotic Gene Expression > TEST 1 REVIEW > Flashcards

TEST 1 REVIEW Flashcards

1
Q

types of repetitive sequences

A

tandem and interspersed sequences

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2
Q

types of tandemly repeated sequences

A

satellite DNA, minisatellite DNA, microsatellite DNA

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3
Q

types of interspersed repeated sequences

A

transposons, MITEs, SINEs, LINEs

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4
Q

satellite DNA

A

large tandem arrays reiterated millions of times (10’s-100’s bp in size), usually AT rich e.g. centromeres

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5
Q

minisatellite DNA

A

repeat units up to 25 bp in length, clustered in 20kb groups, e.g. telomeres are TTAGGG x100’s

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6
Q

microsatellite DNA

A

clusters of 150 bp of repeated units of 2-6 bp, located in euchromatin and generated via slippage, polymorphisms used in genetic profiling

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7
Q

transposons

A

contain single gene encoding transposase flanked by inverted terminal repeats, ~1-2 kb

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8
Q

MITEs

A

mini inverted repeat transposable elements – can regulate gene expression by acting as cis-regulatory motifs, palindromic and contain ITRs

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9
Q

SINEs

A

less than 500 bp, related to retroviruses but do not contain LTRs, transpose through RNA intermediate

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10
Q

LINEs

A

more than 5 kb, appear to be remnants of retroviruses, contain 2 ORFs encoding 2 proteins (1 being reverse transcriptase), often have degenerate 5’ end

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11
Q

Mechanisms of tandem repetitive sequence generation

A

replication slippage, unequal crossing over, unequal crossing over, unequal sister chromatid exchange, errors in single strand break repair

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12
Q

replication slippage

A

generates diversity in short repeats – deletion error correction leads to addition of extra bases, implicated in trinucleotide repeat expansion diseases

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13
Q

unequal crossing over

A

for longer repeats – unequal crossing of pairs of homologous chromosomes

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14
Q

unequal sister chromatid exchange

A

one gets both homologous sequences

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15
Q

errors in single strand break repair

A

during DNA replication

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16
Q

mechanisms of interspersed repeated sequence generation

A

transposons, retrotransposons, LINEs, SINEs

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17
Q

transposons interspersed repeated sequence generation

A

transposase makes blunt end cuts in transposon and sticky end cuts in target DNA and ligates transposon in place

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18
Q

retrotransposons interspersed repeated sequence generation

A

transcribed by RNAPII, processed into mRNA, then reverse transcribed into dsDNA somewhere else in the genome

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19
Q

LINE generation

A

ORF protein (attached to LINE RNA) nicks genome at AT rich site, reverse transcription primed by chromosomal DNA is completed by ORF protein, insertion completed by cellular enzymes e.g. L1 - Promoter sequences for LINEs direct RNAPII-dependent transcription

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20
Q

SINE generation

A

5’ end contains RNAPIII promoter, does not code for transposase or integrase as it hijacks LINE machinery e.g. Alu

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21
Q

techniques to measure sequence copy number

A

PCR, FISH, DNA microarrays

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22
Q

PCR

A

use primers to amplify alleles in DNA sample, run on electrophoretic gel, determine size – tandem repeats

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23
Q

FISH

A

fluorescence in situ hybridization – fluorescently labeled DNA probes amplified by PCR are fixed to cells/tissue and observed under fluorescent microscope, used to detect interspersed repeats, measure copy number

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24
Q

DNA microarrays for detection of deletions and duplications

A

Immobilize probe sequence on chip, extract and shear DNA sample, generate labeled genomic fragments in vitro, hybridize to array, measure intensity with scanner, Comparative genome hybridization - comparing individual to reference

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25
Q

stabilizing selection

A

both copies retain function until they subfunctionalize

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26
Q

selective pressure on both copies

A

genes stay similar

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27
Q

selective pressure on one copy

A

either one copy will degrade or one will acquire a new function

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28
Q

globin loci at different stages of development

A

Fetal – a2g2, embryonic – a2e2, adult – a2b2, Theory that each globin is derived from one copy and subfunctionalized to optimize oxygen binding

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29
Q

Dlx genes

A

many species have different copies on different chromosomes, the sequences are conserved but expression profiles now vary considerably

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30
Q

Model mechanisms of divergent gene expression after duplication event

A

DDC model, whole genome duplication, horizontal gene transfer, de novo creation from random transcription, expression first model, ORF first model

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31
Q

DDC model

A

changes in expression due to complementary loss of region specific regulatory sequences (a type of subfunctionalization), decreased breadth and increased specificity in expression

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32
Q

example of DDC model

A

gsb and prd are parologous genes in drosophila, in gsb knockout, prd can rescue the phenotype if expressed using gsb promoter

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33
Q

whole genome duplication

A

aberrant meiosis at prophase two and fusion of diploid gametes

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34
Q

expression first model

A

stop codons present in genes then mutates to form ORF

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35
Q

ORF first model

A

ORF present but only later gains promoter to attract TF

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36
Q

chromatin organization in the nucleus

A

euchromatin surrounds heterochromatin all over nucleus, with nucleolus in centre, rich protein based matrix facilitates protein-DNA interactions

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37
Q

chromosome territories

A

chromosomes maintain spatially defined volume, radially organized with some in inner circle and some at periphery but positions are not absolute in all cells

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38
Q

chromosome territories experiment

A

Photobleaching experiment showed that fluorescently labeled genes in each hemisphere generally retained localization after mitosis

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39
Q

organization of heterochromatin

A

contains satellite DNA, transposable elements, and some functional genes

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40
Q

position effect variegation

A

heterochromatic regions can produce variegated expression of euchromatic genes when the two are juxtaposed

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41
Q

constitutive heterochromatin

A

permanently condensed regions e.g. centromeres and many regions of Y chromosome

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42
Q

facultative heterochromatin

A

non-permanently condensed regions associated with inactive genes (due to cell specialization)

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43
Q

SAR

A

scaffold attachment region (attached to scaffold protein, AT rich)

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44
Q

MAR

A

matrix associated region (AT rich)

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45
Q

functional chromatin domains defined by

A

DNase I sensitivity or being bound by insulator sequences

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46
Q

3C

A

technique to study genome organization and structure - chromosome conformation capture – done by looping a segment of DNA, cross linking the portion that connects the loop, digesting the sequence, reversing the cross linkage and amplifying/determining the sequence using PCR

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47
Q

insulators

A

maintain independence of a functional domain (1-2 kb) by blocking interactions between enhancers and promoter and often include binding domain for CTCF-zinc finger protein (leading to nucleoprotein complex assembly), recruit chromatin modifying enzymes, protect against PEV

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48
Q

nucleosomes

A

fundamental unit of chromatin containing 2 molecules each of H2A, H2B, H3, H4, H2A.Z; centromeres contain H3 variant cenH3 leading to tetrameric histones (hemisomes)

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49
Q

methods of next generation sequencing

A

ilumina, transcriptomics, DNA microarrays, DNA chips

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50
Q

ilumina

A

sequencing by synthesis approach – randomly fragment DNA sample and ligate adaptors to either end of the fragments, bind ss fragments randomly to inside surface of the flow channels, bridge amplification; use modified bases that emit light

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51
Q

transcriptomics

A

study of full set of mRNAs present in a cell at any given time or conditions (main goals identifying mRNAs present and their relative abundance)

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52
Q

technique to study transcriptomics

A

RNA-seq

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53
Q

RNA-seq

A

convert all mRNA into cDNA and sequence that, can be done using DNA hybridization methods

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54
Q

DNA microarrays for NGS

A

ability to monitor expression of thousands of genes simultaneously by hybridization using glass or nylon surfaces spotted with DNA molecules

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55
Q

DNA chip

A

same process as microarray but using glass or silicon wafer spotted with an array of immobilized oligos with segments matching each gene

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56
Q

cluster analysis

A

distinguishing complex differences in gene expression, genes that display similar expression profiles under different temporal or environmental conditions may have related functions, can be grouped by a method of hierarchical clustering where expression intensity is assigned a value that indicates degree of relatedness between the expression levels

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57
Q

complications with transcriptomic analysis

A

o Transcripts from more than one gene may hybridize to the same probe
o Different mRNAs from the same gene are difficult to distinguish
o Only tells abundance in mRNA which is not necessarily indicative of rate of transcription or rate of transcript degradation or changes in protein levels

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58
Q

ChIP-chip process

A

isolate and shear chromatin, add antibody specific for acetylated N-terminal tail, immunoprescipitate and release and amplify DNA, fluorescently label, hybridize to chip

59
Q

ChIP-seq process

A

isolate and shear chromatin, add antibody specific for acetylated N-terminal tail, immunoprescipitate and release and amplify DNA, fractionate, do NGS

60
Q

CRISPR-Cas9 editing

A

RNA guided double stranded DNA cleavage using ssDNA oligo for repair

61
Q

known uses for CRISPR-Cas9

A
  • Indel creation/repair
  • Gene insertion or replacement
  • Large deletion or rearrangement
  • Gene activation
  • Chromatin or DNA modifications
  • Imaging location of genomic locus
62
Q

Class 1 chromatin remodelling enzymes

A

covalent modification of histones, modifications that indirectly regulate chromatin structure through recruitment of chromatin-associated proteins e.g. histone tail modifications (HAT, HMT, HDAC, HDM)

63
Q

class 2 chromatin remodelling enzymes

A

ATP dependent multiprotein remodeling complexes, directly overcoming repressive nucleosomes, use energy from ATP hydrolysis to alter physical properties of nucleosomes so DNA is more accessible (nucleosome displacement) – all have highly conserved helicase-like ATPase domain

64
Q

histones

A

o Globular domain that interacts with other histones and DNA

o Flexible N- and C-terminal tail regions that act as substrate for various post translational modifications

65
Q

histone acetylation regulation in yeast

A

DNA specific element binds to TF which binds HAT which adds acetyl OR repressor binds HDAC to remove acetyl

66
Q

ATP dependent chromatin remodeling complexes

A

SWI/SNF, CHD

67
Q

SWI/SNF

A

contains bromodomain that binds acetylated histone

68
Q

CHD

A

contains chromodomain that binds methylated histone

69
Q

nucleosome remodelling

A

change in structure, sliding – displacement along DNA, transfer – removing and transferring to non-adjacent region of DNA

70
Q

DNA hypersensitive sites (DHS)

A

DNase I assays demonstrate that sensitive sites in DNA tend to be upstream of promoter sites

71
Q

drosophila Hsp70

A

after heat shock, hsp70 promoter is remodeled by a HAT complex to create a new DHS

72
Q

mechanisms of nucleosome dependent transcription in yeast

A

TFs bind and decondense chromatin, enable acetylation of histones, then acetylated histones attract remodeling complexes (SWI/SNF) which remove H2A.Z (histone eviction) and transfer them to chaperone proteins which will recycle them into new histones

73
Q

regulation of histones during transcription

A

downstream of loose chromatin, nucleosome occupancy is maintained to prevent inappropriate transcriptional initiation, regions that have already been transcribed are quickly deacetylated because Set2 protein of RNAPII will methylate H3K36 which is recognized by Rpd3 decetylase complex

74
Q

promoters

A

integrates all of the regulatory inputs in order to cause transcription to occur, minimum region required for docking of transcription machinery

75
Q

core promoter

A

surrounds transcription start site (80 bp) containing features that are sufficient for recognition of transcriptional machinery

76
Q

proximal promoter

A

includes additional regulatory information (~300 bp upstream of the core promoter)

77
Q

experimental characterization of promoters

A

5’ end deletion experiments, mutation scans, DNase footprinting

78
Q

5’ end deletion experiments

A

help define the minimal promoter that is required for transcription

79
Q

mutation scans

A

making sequential deletions along regulatory regions and measure level of expression of reporter – for finding precise elements within the promoter that are required

80
Q

DNase footprinting

A

take labeled fragments of promoter, digest using DNase I, which will cut all regions of DNA besides where any proteins are bound

81
Q

consensus sequences

A

short regulatory DNA elements are highly conserved across eukaryotic species, but there is no universal core promoter – genes contain many combinations of promoter elements within their core promoters

82
Q

base pair substitution analyses

A

comparing consensus sequences help define the precise sequence of each functional element in the promoter

83
Q

types of promoter sequences

A

TATA box, Inr element, downstream promoter element

84
Q

TATA box

A

usually 25-30 bp upstream of TSS, typically recognized by the binding subunit of TFIID

85
Q

Inr element

A

pyrimidine, any nucleotide, usually within 10 bp of TSS, can be identified in promoters that may or may not have TATA boxes, >50% of promoters in all animals

86
Q

downstream promoter element

A

– usually identified in TATA-less promoters (DPE+Inr common), positioned 50 bp downstream of TSS, not recognized by TBP of TFIID, 40-50% of animal promoters

87
Q

TF domains

A

functional and transcription activation

88
Q

functional TF domains

A

DNA binding domain, transcription activation domain, other protein interaction domain

89
Q

transcription activation TF domain

A

acidic, glutamine rich, proline rich, beta sheet domains

90
Q

methods of protein DNA interactions

A

EMSA, SELEX, co-transfection, ChIP

91
Q

EMSA

A

electro mobility shift assay, run fragments on a gel then mix fragments with cell bits that you suspect contain proteins then if it runs at a different position on the gel a protein has bound

92
Q

SELEX

A

gene regulatory protein of unknown specificity added tot large pool of short DNA double helices, then run on a gel to determine which fragment the protein bound to, then sequence that fragment

93
Q

co-transfection

A

in vivo - add two plasmids to yeast at the same time, one with DNA elements and one with protein elements and see if they work together (requires that the cell is unable to be incapable of plasmid endogenous transcription of the reporter gene plasmid

94
Q

classes of DNA binding domains

A

basic helix loop helix, leucine zipper, C2H2 zinc finger

95
Q

basic helix loop helix

A

specific dimerization region of the domain, basic residues make favorable interactions with negative DNA, helix fits in to the major groove

96
Q

leucine zipper

A

basic region responsible for binding, formed by two polypeptides, each one is an alpha helix with leucine spaced 7 residues apart so they all face inside the helix, two monomers form a parallel coiled coil

97
Q

C2H2 zinc finger

A

anti parallel beta strand connected to an alpha helix by a short loop, two cysteines in the beta strand and two histidines in the alpha helix coordinate the zinc ion, the alpha helix interacts with the bases of DNA, the beta strand binds to DNA backbone and positions the recognition helix for optimal interaction

98
Q

nuclear hormone receptors

A

contain DNA binding domain and ligand binding domain, and depending on how they dimerize will make different types of binding specificties

99
Q

transcriptional repression

A

competition, inhibition, direct repression, indirect repression

100
Q

transcriptional repression - competition

A

repressor can inhibit binding of an activator to a gene by binding to overlapping DNA sequences – short range

101
Q

transcriptional repression - inhibition

A

repressor can bind to a separate DNA site close to/or actually with the activator and prevent the activator from interacting with the other components – short range

102
Q

direct transcriptional repression

A

repressor binds to basal transcriptional machinery directly (independently of the activator) – short or long range

103
Q

indirect transcriptional repression

A

repressor recruits chromatin modifying factors (E.g. HDACs) – short range or long range

104
Q

transcriptional repressors recruiting PRCs process

A

repressors have DNA binding domain and repressor domain which interacts with PRC which transfers methyl groups to the histone tails which signals PRC1 to recognize histone tails which creates very tightly packed repressor complex that stays on chromosome turning things off

105
Q

Domains of the Polycomb repressor complex

A

E(z), Eed, Pc, HMT

106
Q

E(z) domain of PRC

A

subunit contains Set domain with HMT activity

107
Q

Eed domain of PRC

A

critical repressor binding component of PRC2

108
Q

Pc domain of PRC

A

contains chromodomains

109
Q

HMT domain of PRC

A

other histone methyltransferases that maintain methylation even after repressors are no longer expressed

110
Q

Enhancers

A

segments of DNA containing multiple TF binding elements (usually non-coding DNA), enhances transcription from a gene containing a core promoter, variable in size, position independent, usually recognized by activators and act to recruit chromain modifying enzymes

111
Q

silencers

A

similar features to enhancers except that silencers repress transcription, sequences are recognized by repressors and act to recruit chromatin modifying enzymes

112
Q

cis-regulatory modules

A

contain a variety of elements that can activate or repress transcription, receive complex combinatorial inputs and results in a functionally integrated response

113
Q

example of cis-regulatory modules

A

mRNA expression pattern of eve - tested using in vivo transgenic analyses in embryos; modular because they work independently, there is a different one for different stripes

114
Q

locus control region

A

how enhancers meet promoters, DNA looping into 3D conformations, may have binding sites for looping proteins that hold together the conformational changes

115
Q

cis-determinant

A

carried on same chromosome, linked to the same gene whose expression they affect, e.g. enhancers, silencers, binding sites, promoters

116
Q

trans-determinant

A

encoded in other places in the genome e.g. HDACs, HATs, TFs

117
Q

cis-regulatory code

A

a particular combination of TF binding sites codes for gene expression

118
Q

functional conservation without sequence conservation

A

accounted for by small changes in TF binding sequences, rearrangement of the order of elements within these larger enhancers or other minute changes that are not detected using current computational methods

119
Q

TBP component of TFIID

A

recognition of TATA box and possibly Inr element, forms a platform for TFIIB binding

120
Q

TAF component of TFIID

A

recognition of core promoter, regulation of TBP binding

121
Q

TFIIA

A

stabilizes TBP and TAF binding

122
Q

TFIIB

A

intermediate in recruitment of RNAPII, influences selection of TSS

123
Q

TFIIF

A

recruitment of RNAPII, interaction with non-template strand

124
Q

TFIIE

A

intermediate in recruitment of TFIIH, modulates activities of TFIIH

125
Q

TFIIH

A

helices activity responsible for the transition from closed to open promoter complex, possibly influences promoter clearance by phosphorylation of CTD of RNAPII

126
Q

PIC assembly sequence

A

o TFIID (TBP and TAF 1-13) recognizes TATA box, Inr, and DPE with TBP subunit which binds to the promoter element, interacts with the minor groove of DNA and bends helix, facilitating TFIIB attachment
o TFIIB adds to TFIID
o TFIIF/RNAPII complex is positioned on top of TSS
o TFIIE binds to create TFIIH docking site
o TFIIH binds and uses ATP hydrolytic helicase activity to unwind DNA, once transcription starts, most components are released but TBP and other TFIID subunits remain for quick facilitation of reinitiation

127
Q

mediator complex

A

25-30 separate subunit proteins also highly conserved, stimulates basal RNAPII transcription in vitro and associates with RNAPII to create a stable holoenzyme, activator elements bound to enhancer elements facilitate activity of general TFs by using mediator as bridge, facilitates multiple rounds of transcription from on initial PIC

128
Q

example of mediator subunit activator specificity

A

transcriptional activators ELK1 and I1A interact with Med23 at Egr1 but when Med23 is mutated the mediator complex still assembles and acts normally except for Egr1

129
Q

mediator, Gal10 and UAS in yeast

A

mediator could interact with the Gal10 while bound to UAS in the absence of functional core promoters and a mutation preventing PIC assembly

130
Q

Covalent modifications that occur at initiation of transcription

A

phosphorylation of CTD of large subunit of RNAPII, methylation of H3 lysine 4 by Set1 complex

131
Q

CTD of RNAPII

A

composed of tandem repeats of a conserved heptad AA sequence 52 times in vertebrates and 26 times in yeast – TFIIH subunit phosphorylates S5 to recruit capping factors in initiation and P-TEFb phosphorylates S2 in elongation

132
Q

classes of TSS in mammalian promoters

A

short and broad

133
Q

RNAPII elongation

A

once transcription is initiated, most TFs are released and replaced by TFIIS, Spt5 and other elongation factors involved in RNA processing – perform functions once recruited to phosphorylated CTD

134
Q

formation of the 5’ cap

A

RNA 5’ triphosphatase, guanyliltransferase, guanine 7-methyltransferase

135
Q

functions of the 5’ cap

A

ensures proper exit to cytosol through binding the CBC, prevents 5’-3’ exonuclease digestion, serves as docking site for translational machinery

136
Q

TFIIS

A

limits length of time RNAPII pauses during transcription, helps RNAPII proofread the transcript, Aids in NTP removal, stimulating RNase activity of RNAPII to remove misincorporated NTPs

137
Q

N-TEF

A

ATP analog of DRB inhibits transcription elongation so that RNAPII comes under control of N-TEF resulting in a trapped complex near the promoter

138
Q

NELF, DSIB

A

In vitro experiments show NELF and DSIF only work combined to slow elongation, and could block TFIIS activity so that RNAPII stays paused

139
Q

P-TEFb

A

phosphorylates the Spt5 subunit of DSIF causing NELF release, phosphorylates S2 of CTD which recruits RNA processing factors during elongation

140
Q

P-TEFb regulation

A

Regulated by autophosphorylation of Cdk9 C-term, T-loop phosphorylation by Cdk activating kinase, ubiquitilation of cyclin T1 by ubiquitin ligase Skp2, recruited by specific TFs or chromatin remodelling complexes

141
Q

HEXIM 1/2, 7SK

A

P-TEFb inhibitors

142
Q

tat locus

A

gene encoding HIV sequence specific RNA binding protein

143
Q

5’ TAR of HIV transcript

A

has sequences recognized by tat and cellular cyclin t, which positions and activates CDK9 to CTD of RNAPII, allowing efficient transcription elongation

144
Q

TAR + tat

A

recruits P-TEFb, can overcome premature termination of transcription by N-TEF

Eukaryotic Gene Expression (1 decks)

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