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L 211 > exam 2 (eukaryote transcription) > Flashcards

exam 2 (eukaryote transcription) Flashcards

1
Q

regulation of gene expression is done by

A

promoters

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2
Q

regulation of transcription is done by

A

DNA binding proteins

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3
Q

whats the difference between prokaryotic and eukaryotic transcription?

A

more complicated with multiple RNAPs, more initiation factors, and chromatin

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4
Q

RNAP1 is associated with

A

rRNA

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5
Q

RNAP2 is associated with

A

mRNA, IncRNA, miRNA

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6
Q

what are introns

A

protein non-coding regions in genes

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7
Q

what are exons

A

protein coding regions in genes

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8
Q

what are the eukaryotic core promoters

A

BREs: TFIIB response elements
TATA box
Inr: initiator element
DPE: downstream promoter element

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9
Q

how does eukaryotic RNAPii differ from bacterial RNAP

A

eukaryotic RNAPii holoenzymes can’t bind promoters wihtout help

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10
Q

what occurs with the preinitiation complex (PIC)

A

RNAPii is recruited to promoters, which contains general transcription factors

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11
Q

what is the TATA-binding protein and its function

A

subunit of TFIID
binds TATA boxes to initiate PIC formation
(only ~10% of promoters have TATA boxes)

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12
Q

what is TFIID

A
  • contains TBP and 13 associated factors
  • contacts Inr and DPE
  • structure to support PIC assembly
  • integrates regulatory signals
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13
Q

TFIIH function

A
  • helps unwind DNA to form the open complex (acts as helicase)
  • it phosphorylates the RNAPii CTD
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14
Q

what is the other promoter on the gene that is upstream of the core promoter

A

regulatory promoter

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15
Q

whats the difference in the two eukaryotic gene promoters

A

regulatory promoters: has binding sites for specific activators
core promoters: bounded by GTFs

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16
Q

how is the strength and the contribution of a promoter tested?

A

reporter assays

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17
Q

common reporters

A

Luciferase
green fluorescent protein (GFP)
lacZ

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18
Q

what is the role of a reporter assay

A

bonds to a promoter in a gene, it is transcribed and translated, and is created into a reporter protein

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19
Q

what are enhancers

A

DNA regulatory element that activates transcription to higher levels, by providing a binding site to be looped in relation to the target promoter gene

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20
Q

for example, if SV40 was added to a eukaryotic gene, what would occur?

A

transcription would increase 100-fold

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21
Q

at what distance does a enhancer need to be at, and what direction did it move

A

can occur over a decently far distance, and can work in forward or backwards direction

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22
Q

decsribe enhancer properties

A

distance independent:
can occur over a decently far distance, 1 kb
orientation independent:
can work in forward or backwards direction

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23
Q

how are complex enhancers created

A

transcription factors bind to numerous enhancers to form a complex code

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24
Q

how are enhancers and promoters similar

A

contain specific DNA sequences bound by various proteins

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25
Q

functions of insulators

A

block enhancer activity

create a boundary between euchromatin and heterochromatin

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26
Q

protein CTCF mediates which function

A

it is the bridge of the insulator

27
Q

what are the two domains an activator must have

A

DNA-binding domain: makes contact with specific DNA sequences in promoters and enhancers
Activation domain: interacts with various components of the transcription machinery

28
Q

what bonds to both the enhancer and regulatory promoter

A

DNA binding domain and activation domain, together creating the activator proteins

29
Q

how is transcriptional activations isolated

A

protein extract and specific DNA sequence that is bonded to beads are mixed. Non-specific DNA is added to complete any proteins that bind DNA nonspecifically. Salt is added to release proteins specifically bounded to the beads. Pure regulatory protein is released and beaded DNA remains

30
Q

how are activators used to screen for proteins that interact with one another

A

2 hybrid assay

31
Q

expression of the normal activator leads to…

A

normal transcription of the reporter gene

32
Q

expression of the bait-DNA binding domain fusion alone leads to

A

no reporter expression

33
Q

expression of the prey-activation domain fusion leads to

A

no reporter expression

34
Q

expression of the bait and prey fusions (assuming they are 2 interacting proteins) leads to

A

robust reporter expression

35
Q

how are activators classified

A

by the structure of their DNA bonding domains

36
Q

what kind of bonds do activators and DNA have

A

hydrogen bonds
ionic bonds
hydrophobic interactions
NOT COVALENT

37
Q

describe helix-turn-helix (HTH) model

A

semicircular, fitting in the major groove of DNA complex

helix 3 is hydrophilic

38
Q

why do most HTH sequence specific DNA binding proteins bond the major groove

A

more features of the bases are exposed

39
Q

the HTH recognizes what sequence

A

5’ AATA 3’

40
Q

function of zinc fingers (ZFs)

A

many different types that use cysteine and hstidine residues to coordinate zinc ions and properly fold the domain

41
Q

describe the ZF of Gal4

A

a yeast activator that uses 6 cysteines to coordinate 2 zinc ions
Zn2Cys6ZF

42
Q

what is Gal4

A
  • dimer (must be to operate)
  • stabilized by coiled coil
  • uses Zn2Cys6ZF to make specific contacts with major groove, spaced 17bp apart
43
Q

what is Gcn4 and its function

A
  • dimer
  • stabilized by coiled coil
  • forms scissor-like structure that makes hydrogen bonds with specific bases and ionic bonds with oxygen atoms in the backbone
44
Q

what is unique about the connection of the mediator complex

A

there is a bridge between enhancer and regulatory promoter, there is no physical connection

45
Q

how many subunits does the mediator complex have

A

25

46
Q

what is the function of the mediator head

A

contacts the PIC

47
Q

whats the function of the mediator middle

A

contacts the various other factors and is a structural support

48
Q

what is the function of the mediator tail

A

contacts TFs bound to the the enhancer

49
Q

how does mediator of yeast and mammals compare

A

mammals have a lot more subunits in the mediator

50
Q

in bacteria how does RNAP get the energy it needs in order to escape the promoter

A

scrunching

51
Q

in eukaryotes, how does RNAP get the energy it needs in order to escape the promoter

A

scrunching, in addition to new mechanisms

52
Q

what is the largest subunit in the RNAPii? and what does it consist of?

A

Rpb1

consists of repeats of AA sequence: YSPTSPS

53
Q

how many of the Rpb1 residues can be phosphorylated

A

5/7

54
Q

how is serine 5 phosphorylated? and its purpose

A

by TFIIH, during initiation

enables RNAPII to break free of the PIC

55
Q

when are serines 2 and 7 phosphorylated

A

during elongation

56
Q

RNAPII is regulated by what factors

A

NELF and DSIF

57
Q

RNAPII is released by

A

P-TEFb

58
Q

where does RNAP pausing occur

A

in a window downstream of where the start of transcription begins (Inr and exons)

59
Q

why does RNAPII pause

A

balances genes for rapid synchronized activation by preloading RNAPII

60
Q

describe elongation

A

RNA strand reads off of the DNA template strand, in the RNAP to match the complementary strand (only t is replaced with u)

61
Q

Pausing is regulated by

A

NELF, DSIF, P-TEFb

62
Q

why does RNAPII stall

A

physical impediments to progress, could be error correction, not enough energy etc

63
Q

what is a nucleosomal role in transcription

A

it prevents it from progressing, by putting up a substantial energy barrier to RNAPII

64
Q

how does RNAPII get through the nucleosomal DNA

A

histone variant H2A.Z helps

L 211 (12 decks)

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