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Flash Cards > molec exam 3 10-13 > Flashcards

molec exam 3 10-13 Flashcards

1
Q

There are 2 RNA polym. inside of eukaryotic nuclei:

A

-one to transcribe major ribosomal RNA genes
-one or more to transcribe the rest of the nuclear genes

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2
Q

Why are ribosomal genes different from other nuclear genes?

A

-different bases/nucleotides than nuclear genes
-unusually repetitive
-found in the nucleolus

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3
Q

Eukaryotic nuclei contain ___ RNA polymerases that can be seperated by _____

A

3, ion exchange chromatography

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4
Q

RNA polymerase 1

A

found in nucleolus, likely to transcribe rRNA genes

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5
Q

RNA polymerase 2 and 3

A

found in nucleoplasm

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6
Q

micro RNA profile …

A

changes from person to person based on epigenetics, or a persons environment

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7
Q

RNA polymerase 1 synthesizes

A

large rRNA precursor, for 28s, 18s, 5.8s rRNAs

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8
Q

RNA polymerase 2 synthesizes

A

hnRNAS (form mRNA), snRNAs, miRNAS (micro)

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9
Q

RNA polymerase 3 synthesizes the precursor for…

A

tRNA

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10
Q

RPB is …

A

RNA polymerase 2

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11
Q

RNA poly 2 has __ subunits

A

12

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12
Q

RPB _ and _ always function together

A

4 and 7

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13
Q

yeast gene orthologs

A

RPB1, RPB2, RPB3

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14
Q

RPB1

A

beta’ ortholog

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15
Q

RPB2

A

beta ortholog

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16
Q

RPB3

A

alpha ortholog

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17
Q

what is hRPB1,2,3…

A

the human version of yeast RPB

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18
Q

Which are the common RPB subunits, common in all three polymerases?

A

hRPB 5, 6, 8, 10, 12
-little is known about their function.
-likely help transcription process.

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19
Q

Epitome tagging purpose

A

helps determine whether a polypeptide is a subunit or co purify with the polymerase.

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20
Q

Epitome tagging mechanism

A

-add extra domain to ONE subunit of RNA poly, with other subunits not changing
-immuno-precipitate with antibody directed against epitope
-denature with SDS detergent, and seperate via electrophoretic gel

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21
Q

RPB’s _______ are REQUIRED for enzyme activity

A

1,2,3

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22
Q

RPB 1, 2, 3

A

-homologous to b, b’, and a subunits
-RPB 1 and b’: these bind DNA
-RPB2 and b: at or near the nucleotide joining active site

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23
Q

What are the similarities between RPB3 and alpha subunit?

A

-a 20 AA subunit very similar
-2 subunits are the same size w/ same stoich.
-2 monomers per holoenzyme
-so, RPB3 is homologous to alpha

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24
Q

What are the two forms of RPB1?

A

-one can bind to the promote (llb)
-one can transcribe and elongate (llo)

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25
Q

RPB1 gene product is subunit ___

A

lla.
-lla is the primary product in yeast
-lla is converted to llb by proteolytic removal of the CTD
-converts to llo by phosphorylating 2 serine in the repeating heptad of CTD
-enzyme with lla binds to the promoter
-enzyme with llo is involved in the transcript elongation

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26
Q

CTD

A

carboxyl terminal domain.
-7 peptide repeated over and over

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27
Q

Which AA’s undergo phosphorylation>

A

serine
threorine
tyrosine

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28
Q

RPB 4 and 7 help ________

A

-initiation.
-with rpb 4 and 7, clamp is forced shut
-initiation occurs w the clamp shut, and promoter DNA melts to let template DNA enter active site
-rpb 4 and 7 extend dock region of poly, making it easier for transcription factors to bind

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29
Q

RPB 7

A

binds to nascent RNA, directing it to CTD

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30
Q

The three RNA polys differ by :

A

different structures
transcribe different genes
recognize different promoters

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31
Q

RNA poly 2 recognizes class ___ promoters

A
  1. Contains:
    core promoter- attracts gtf’s and rna poly at basal level, sets transcript start site, and direction of transcription
    proximal promoter- enhances transcription. helps attract gtf’s and rna poly, and includes promoter elements upstream of the transcription start site
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32
Q

Core promoter is modular- it can contain any combo of THESE elements:

A

-TATA box (31 to 36)
-TFIIB recognition (BRE, 37 to 32)
-Initiator (INR, 2 to 4, overlaps w transcript start site)
-downstream promotor element (DPE, 28 to 32)
-downstream core element (DCE, 3 parts)
-motif ten element (MTE 18, to 27)

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33
Q

TATA box

A

-TATAAA
-similar to prokaryotic 10 boc, centered at -28
-only found in specialized genes, like keratin skin cells or hemoglobin
-promoters are found w no TATA box

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34
Q

TATA-less promoters

A

-have DPEs
-and are found in 2 classes of genes:
a) housekeeping genes: active in all cells, they control common biochem pathways
b) developmentally regulated genes: control development of fruit fly or immune system in mammals

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35
Q

upstream promoter elements

A

-found upstream of class 2 core promoter elements
-differ from core promoters in binding to gene-specific transcription factors
-UPE can be orientation dependent, and position dependent- have to be close by.

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36
Q

enhancers and silencers

A

-position and orientation dependent DNA elements
-rely on tissue specific DNA binding proteins for their activities
-some DNA elements can act either as enhancer or silencer, depending on what is bound to it: like thyroid hormone response element> when bound , enhancer. when not bound, silencer

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37
Q

enhancers

A

act through the proteins bound to them.
-these proteins are either transcription factors, enhancer binding proteins, or activators
-proteins appear to STIM transcription by interacting with general transcription factors at the promoter
-promote pre-initation complex formation
-usually found upstream of the promoter they control

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38
Q

silencers

A

act at a distance just like the enhancers to modulate transcription
-INHIBITS transcription
-cause chromatin to coil up, into an inaccessible form to prevent further transcription

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39
Q

eukaryotic rna poly

A

-INCAPABLE of binding to promoters by themselves
-rely on transcription factors to direct them:
general transcription factors and gene specific transcription factors (AKA activators)

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40
Q

GTF’s combine with rna poly to form a _______ complex.

A

pre-initiation
-initiates transcript when nucleotides are available
-tight binding forms an open promoter complex with DNA at the transcript start site that has melted

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41
Q

Class 2 preinitation complex

A

contains:
-rna poly 2, six gtf’s:
TFII A, B, D, E, F, H
-these tf’s and poly form the preinitiation complex in a specific order

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42
Q

Transcription factors bind to class 2 promoters in this order:

A

-DA complex: TFIID w help from TFIIA binds to TATA box
-DAB complex: TFIIB binds DA complex.
-DABPolF complex: TFIIF helps RNA poly bind to a region from 34 to 17
-DABPolFEH complex: TFIIE and then TFIIH bind to form complete preinitiation complex

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43
Q

TFIID contains these subunits:

A

TATA box binding protein (TBP)
-evolutionarily conserved, binds to minor groove of TATA box. saddle shaped TBP lines up w DNA, bottom of saddle forces minor groove open, TATA box is now bent

TBP associated factors (TAFs)
specific for Class2. occurs when theres no TATA box.

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44
Q

TBP (tata binding) mutant cell extracts are deficient in …
TBP is a ….

A

-transcript of class 1, 2, and 3 genes
-universal transcription factor required by poly 1, 2, and 3

45
Q

TBP associated factors (TAFs)

A

-13 TAFs are associated w class 2 preiniation complexes
-named according to size, large to small
-interact w core promoter elements and gene specific transcription factors
-when TBP is attached, it will extend the binding of TFIID beyond the TATA box

46
Q

Roles of TAF’s:

A

TAF1: helps TFIID bind to initiator
TAF1 has 2 enzymatic activities: histone acetyltransferase and protein kinase
TAF2: helps TFIID bind to DPE
TAF’s enable TBP to bind to TATA-less promoters, that have elements like a GC box.
-different combos of TAF’s is needed for responding to different activators

47
Q

TFIIH

A

last general transcription factor to join preinitiation complex
-it contains 9 subunits
-2 major roles in transcription initiaition:
-phosphorylates CTD of RNA poly 2
-unwinds DNA at transcript start site to create transcription bubble

48
Q

phosphorylation of the CTD of RNA poly 2

A

-preinitiation complex is formed w hypo phosphorylated RNA poly 2 (lla)
-TFIIH phophorylates serines 2 and 5 in the heptad repeats (YS2PTS5PS7) in the CTD of the largest RNA poly subunit:
creates the phosphorylated form of the poly enzyme llo, phosphorylation at this site is essential for initiation of transcript

49
Q

minimal initiation complex

A

TFIID, TFIIB, TFIIF, and RNA poly2 form minimal initiation complex at initiator site
-when TFIIH, TFIIE, and ATP are added they allow DNA melting and partial phosphorylation of the CTD of the largest RNA poly subunit
^^^these events allow abortive transcripts to be made, as the transcription stalls at 10.

50
Q

expansion of transcription bubble

A

-energy is provided by ATP
-DNA helicase of TFIIH unwinds DNA
-expansion of the transcription bubble releases stalled polymerase, polymerase clears promoter

51
Q

elongation complex continues elongating the RNA synthesis when

A

polymerase CTD is further phosphorylated by TEFb, NTPs are continuously available
-TBP and TFIIB remain at the promoter
-TFIIE and TFIIH are not needed for elongation, and leave the elongation complex

52
Q

TFIIS

A

-TFIIS stims proofreading
-TFIIS stims RNase activity of the RNA poly, allowing RNA poly to cleave off an incorrect nucleotide and replace it.

53
Q

___________ dictate the starting point and direction of transcription

A

General TF’s.
-by themselves, they will create low level of transcript/basal transcription.
-transcription of many active genes in cells rises above the basal level
-euk cells have extra gene specific TF’s- the activators.
activators bind to enhancers to boost transcription.

54
Q

activators can _____ or ______ transcription by RNA poly 2

A

stim or inhibit.
-activators are made of at least 2 domains:
-dna binding domain
-transcription activation domain
-dimerization domain

55
Q

protein domain

A

independently folded region of protein
-have DNA binding motif
-part of the domain has shape specialized for DNA binding
-most motifs fall into 3 classes: zinc containing modules, homeodomains, bZIP and bHLH

56
Q

zinc containing modules

A

3 kinds, they act as DNA binding motifs:
zinc fingers, zinc modules, modules with 2 zinc ions and 6 cysteines
-all three use one or more Zinc ions to create a shape to fit an alpha helix of the motif into the DNA major groove

57
Q

Homeodomains

A

-contain 60 AA’s
-helix turn helix structure/function
-found in a variety of activators
-identified in homeobox proteins for fruit fly development

58
Q

bZIP and bHLH motifs

A

-highly basic DNA binding motif linked to protein dimerization motifs
-leucine zippers
-helix loop helix

59
Q

transcription activating domains

A

activators have one of these domains, some have more than one:
-acidic domains
-glutamine rich domains
-proline rich domains

60
Q

DNA binding motifs of activators: structure

A

-well defined structures
-x ray crystallog show how these interact w DNA targets
-interaction domains can form dimers or tetramers
-most DNA binding proteins can’t bind DNA in monomer form

61
Q

Three Zinc fingers in a Curved shape

A

zinc finger is made of:
anti parallel beta strand with
2 cysteines, 2 histidines in alpha helix, helix and beta strand are coordinated to a zinc ion

62
Q

A third class of zinc modules are found in ________

A

nuclear receptors.

nuclear receptors are proteins that interact with a variety of endocrine signaling molecules.
-protein+endocrine molecule = complex that functions as an activator, binding to hormone response elements

63
Q

Type 1 Nuclear receptors

A

-reside in cytoplasm, bound to another protein.

when receptors bind to their hormone ligands:
-they release their cytoplasmic protein partners, move to nucleus, bind to enhancers, act as activators

64
Q

Glucocorticoid Receptors: type 1 Nuclear Receptor

A

-DNA binding domain with 2 zinc containing modules
-one module has most DNA binding residues, the other has surface for protein protein interaction to form dimers

65
Q

Type 2 and 3 Nuclear Receptors

A

-Type2 nuclear receptors stay in the nucleus bound to target DNA sites, like thyroid hormone receptors
-without ligands, the receptors repress gene activity, when receptors bind ligands, they activate transcription
-type 3 receptor ligands are not identified “orphan”

66
Q

homeodomain DNA complex

A

-proteins are members of the helix turn helix family of DNA binding proteins
-each homeodomain has 3 alpha helices, 2nd and 3rd forming the helix turn helix motif
-a recognition helix fits into the DNA major groove and makes specific contacts there.
-N terminal arm goes into adjacent minor groove

67
Q

bZIP domains

A
  • proteins dimerize through a leucine zippper
    -puts adjacent basic regions of each monomer in position to embrace DNA target like tongs
68
Q

bHLH domains

A

-proteins dimerize through a helix loop helix motif, allows basic parts of each long helix to grasp the DNA target
-bHLH and bHLH-ZIP domains bind to DNA in the same way, but bHLH-ZIP have extra dimerization due to leucine zippers

69
Q

Bacterial core RNA polymerase is….

A

incapable of initiating meaningful transcription

70
Q

RNA poly holoenzyme can…

A

catalyze basal level transcription
-insufficient at weak promoters
-cells have activators to boost basal transcript to higher level in recruitment process.

71
Q

eukaryotic activators recruit RNA poly to the …

A

promoters.
-they stimulate binding of the GTF’s and RNA poly.
the hypothesis:
-gtf’s cause stepwise build up of preinitiation complex, or gtf’s + proteins are already bound to poly, in a RNA poly holoenzyme

72
Q

__________ must interact to form the preinitiation complex

A

GTF’s

73
Q

Activators and ______ interact

A

GTF’s

74
Q

activators usually interact with one another in activating a gene

A

-individual factors interact to form a protein dimer helping binding to a single DNA target
-specific factors bound to different DNA target sites can collaborate in activating a gene

75
Q

dimerization

A

-increases affinity between activator and its DNA target
-some activators form homodimers but others function as heterodimers

76
Q

insulators

A

-can shield genes from activation by enhancers, enhancer blocks activity
-can shield genes from repression by silencers, barrier activity

77
Q

regulation of transcription factors:

A

-phosphorylation
-ubiquitylation
-sumoylation
-methylation/acetylation

78
Q

phosphorylation

A

phosph. of activators can allow them to interact with coactivtors, to stim transcript

79
Q

ubiquitylation

A

ubiquitylation of tf’s can mark them for destruction by proteolysis and for stimulation of activity
-mono-ubiquitylation especially of some activators can have an activation effect
-poly-ubiquitylation marks these proteins for destruction
-proteins from the 19s regulatory particle of proteasome can stim transcript

80
Q

sumoylation

A

attachment of polypeptide SUMO which can target incorporation into compartments of the nucleus
-SUMO=small ubiquitin related modifier
-101 AA polypeptide
-sumoylation is the addition of one or more copies of SUMO to lysine residues on a protein, process similar to ubiquitylation, but results are different.
-sumoylated activators are targeted to a specific nuclear compartment that keeps them stable

81
Q

methylation/acetylation

A

modulate activity

82
Q

acetylation

A

non histone activators and repressors can be acetylated by histone acetyltransferase (HAT)
-HAT can act on non histone activators and repressors
-can have either pos or neg effects

83
Q

Eukaryotic genes can exist as naked DNA/or DNA molecules only bound to tf’s (t/f)

A

false

84
Q

Chromatin

A

DNA + protein to form chromosomes
-chromatin is variable, and the different versions play an enormous role in chromatin structure/the control of gene expression

85
Q

euk cells have 5 kinds of histones:

A

H1, H2A, H2B, H3, H4

86
Q

histone proteins are not homogenous because…

A

gene reiteration, and posttranslational modification

87
Q

histone structure

A

-abundant proteins whose mass in nuclei nearly equals that of DNA
-positive charge at neutral pH
-well conserved from one species to another
-not single copy-they are repeated many times.
-some copies are identical, some vary
-H4 has only 2 variants ever reported

88
Q

post translational modification of histones

A

-most common mod is acetylation
-at N terminal amino groups and lysine E amino groups
-methylation and phosphorylation also take place at lysine E amino groups
-serine and threonine undergo O phosphorylation
-histone modifications influence chromatin structure and function, and play important role in gene activity

89
Q

nucleosomes

A

chromasomes are long and thin molecules, they will tangle if not properly folded. folding occurs in a few ways, the first order of folding is the Nucleosome.

-nucleosomes have core of histones, and DNA winds around it.
-X ray diffrac has shown strong repeats of structure at 100A intervals
-this corresponds to a string of nucleosome about 110A in diameter

90
Q

histones in nucleosome

A

chemical cross linking in soln:
H3 - H4
H2A - H2B

H3 and H4 exist as a tetramer.
corresponds to 1 histone octamer per 200 bp of DNA
Octamer is composed of 2 each H2A, H2B, H3, H4
1 each H1 bound to octamer

91
Q

chromatin is made of

A

roughly equal mass of DNA and histones.

92
Q

H1 and chromatin

A

treatment of chromatin with trypsin or high salt buffer removes histone H1, leaving chromatin like beads on a string
the beads are nucleosomes.
core histones form a ball with DNA wrapped on outside, DNA on outside minimizes amount of DNA bending.
H1 lies on the outside of the nucleosome

93
Q

Nucleosome structure

A

central H3 H4 core attached to H2A H2B dimers
-grooves on surface define a left hand helical ramp, the path for DNA winding
-DNA winds almost twice around the histone core condensing DNA length by 6 to 7 x.
-core histones contain histone fold

94
Q

histone fold

A

3 alpha helices linked by 2 loops
extended tail of about 28 % core histone mass
tails are unstructured

95
Q

second order of chromatin folding produces a 30nm fiber

A

string of nucleosomes condenses to form the 30nm fiber in a soln of increasing ionic strength
-this condensation results in another 6 to 7 fold condensation of the nucleosome itself
4 nucleosomes condensing into the 30nm fiber form a zig zag

96
Q

solenoid model

A

hollow compact helix

97
Q

high order chromatin folding

A

30nm fibers account for most of the chromatin in a typical interphase nucleus
further folding is needed in structures like the mitotic chromosomes
the series of radial loops is a model known for high order folding

98
Q

histones, especially H1, have a _____ effect on gene activity

A

repressive

99
Q

histones play a predominant role as _____ of ______ and are not just structural

A

regulators, genetic activity

100
Q

histone H1 causes _______ of template activity and core histones

A

further repression

101
Q

H1 repression can be…

A

counteracted by tf’s

Sp1 and GAL4:
anti repressors, prevent histone repression, as well as they can act as transcription activators

102
Q

transcription factors can cause anti repression by:

A

-removing nucleosomes that obscure promoter
-preventing initial nucleosome binding to promoter

^^both actions are forms of nucleosome positioning, activators force nucleosomes to take up positions around promoters

103
Q

histone acetylation

A

occurs in cytoplasm and in nucleus.

cytoplasmic acetylation carried out by histone acetyltransferase B (HAT-B):
prepares histones for incorporation into nucleosomes, and acetyl groups later removed in nucleus

104
Q

nuclear acetylation

A

-catalyzed by HAT-A
-acetylates core histone N terminal tails
-correlates with transcription activation
-coactivators of HAT-A found, may loosen nucleosomes with gene control region
-attracts bromo domain proteins, essential for transcription

105
Q

histone deacetylation

A

transcription repressors: bind to DNA sites and interact w co repressors which bind to histone deacetylases

retinoic acid receptor: binds to retinoic acid response elements (RARE) as a heterodimer with RXR (retinoid x receptor)

in the absence of RA, the RAR-RXR heterodimer inhibits transcription through recruitment of histone deacetylases and nuclear receptor corepressors

106
Q

In the presence of RA, the RAR-RXR heterodimer

A

leaves the HDAC complex and recruits transcriptional coactivators

107
Q

HDAC in transcription repression

A

-assembly of complex brings histone deacetylases close to nucleosomes
-deacetylation of core histones allows histone basic tails to bind to DNA and histones in nearby nucleosomes to inhibit transcription

108
Q

Heterochromatin

A

euchromatin:
extended and open chromatin, potentially active

heterochromatin:
very condensed, DNA is inaccessible
-microscopically appears as clumps in higher eukaryotes
-repressive ability able to silence genes even 3kb away.

Flash Cards (21 decks)

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