lecture 17- transcription in eukaryotes

Question 1

name the RNA polymerases used to synthesize RNA in eukaryotes

Answer 1

RNA pol I, II, III are used for synthesis of different types of RNA (affinity for different promoters)

Question 2

what is required for recognition of promoters in eukaryotes

Answer 2

transcription factors (not polymerases- sigma factors)

Question 3

describe general transcription factors (GTF’s)

Answer 3

GTFs are equivalent to sigma factor of bacteria

TFII(X) used for RNA poly II
TFIII(X) used for RNA poly III
(X) = A, B, C

RNA poly I and III are more selective to promoters- can recognize smaller number of promoters than RNA poly II

Question 4

which RNA polymerase recognizes the most promoters

Answer 4

RNA poly II

Question 5

what TF do all RNA poly’s use?

Answer 5

TBP (TATA-binding protein)
TBP plays a major role in transcription initiation, used for transcription initiation of all genes (presenting or not TATA box)

Question 6

what is the TATA box sequence
how many human genes have TATA box

Answer 6

5’-TATAAA sequence near position -30
only 25% of human genes have TATA box in promoter

Question 7

how is TBP recruited to a gene when there is no TATA box present

Answer 7

recruited to gene by TBP Associated factors (TAFs)

Question 8

describe RNA polymerase I

Answer 8

transcribes ribosomal RNA (rRNA) genes
- rRNA is synthesized in nucleolus, so RNA poly I functions in nucleolus
- rRNA is synthesized on huge 45S RNA and then processed to 5.8S, 18S, and 28S

Question 9

RNA polymerase I recognizes certain sequences in promoters to produce rRNA, describe this

Answer 9

promoter has core sequence and Upstream Control Element (UCE)
- upstream of these is UBF (upstream binding factor), which binds the 2 sequences and recruits RNA poly I and another protein called SL1 (complex of TBP and 3 TAF’s)

Question 10

what does SL1 (selectivity factor) bind concerning promoters for RNA poly I

what happens once all these bind?

Answer 10

UBF’s and poly I

once these bind, transcription can initiate

Question 11

what kind of RNA does polymerase III synthesize?

Answer 11

transcribes tRNA, 5S RNA, small non-coding RNA

Question 12

how many promoters can RNA polymerase III initiate at?

Answer 12

initiates at three types of promoters and requires 2 or 3 TF’s:
1- tRNA: need TFIIIB and TFIIIC
2- 5S rRNA: need TFIIIA, TFIIIB, TFIIIC
3- other small RNA’s: require other specific factors in addition to TFIIIB

Question 13

for RNA poly III, what does the TF TFIIIB include?

Answer 13

TBP

Question 14

describe factors and boxes involved for tRNA promoter for poly III

Answer 14

the promoters present sequences BoxA & BoxB (conserved nucleotides) to transcribe tRNA
- after TFIIIC binds these, recruits TFIIIB which has TBP —> TFIIIC then dissociates and RNA poly III can bind and begin transcription

Question 15

describe factors and boxes involved for 5S rRNA promoter for poly III

Answer 15

BoxA and BoxC in the promoter
- TFIIIC binds BoxA & BoxC, TFIIIA binds BoxA
- this recruits TFIIIB which has TBP — RNA poly III then comes in and transcribes

Question 16

termination of transcription of RNA poly I and III

Answer 16

RNA pol I and RNA pol III terminate at specific terminators (like prokaryotes)

Question 17

describe RNA pol I termination

Answer 17

terminator site located downstream from the pre-rRNA sequence
- requires additional proteins- termination factors that come and release RNA poly I and RNA from template

Question 18

describe RNA pol III termination

Answer 18

terminates transcription at T-rich sequence in DNA template located a short distance from the 3’ end of the mature RNA (so long sequence of adenines in the RNA) , assisted by just a few protein factors

Question 19

what RNA’s does RNA poly II synthesize

Answer 19

mRNA’s, microRNA’s (regulate gene expression), some ncRNA

Question 20

overview of RNA poly II promoters

Answer 20

RNA poly II promoters come in many varieties but share certain sequence features
- RNA poly II promoters always have initiation sequence (Inr) — TATA box and other sequence elements recognized by proteins that bind to polymerase are required for transcription by Poly II
- RNA poly II promoters sometimes include a TFIIB recognition element (BRE), and the downstream promoter element (DPE)

Question 21

RNA polymerase II promoters require several factors for the assembly of the basal transcription apparatus, which are…

Answer 21

RNA poly II, TFIIA, B, D, E, F, H, J

Question 22

describe RNA poly II promoters in little more detail

Answer 22

core promoter: place where basal transcription apparatus assembles
- all RNA poly II promoters are modular and composed of a few elements (cis-acting sites) that will be bound by TFIID in a sequence-specific manner (TF’s are trans-acting)
- only 25% of human genes have TATA box - an upstream element present around -30
- RNA poly II core promoter contains Inr around the startpoint + at least one more element:
- TATA box sequence
- TATA-less promoters must contain at least one other element downstream from Inr: DPE (downstream promoter element) or DCE (downstream core element)

Question 23

RNA POLY II:
building of the initiation complex is sequential and starts with ___ …explain its structure

Answer 23

TFIID
composed of 14 subunits (mostly different TAF’s)
- TAF = TBP-Associated Factor (TAF#)
- different TAF’s have different affinities for different sequence elements that may be present within different core promoters
- TBP binds to promoters ONLY at TATA-box when present

Question 24

RNA POLY II:
building of the initiation complex is sequential and starts with TFIID, how does it bind?

Answer 24

TFIID binds to Inr in TAT-less promoters first via TAF
- other TAFs, depending on the promoter, bind to downstream elements

Question 25

describe the steps of building the initiation complex for RNA poly II

Answer 25

1- once TFIID is bound, the assembly of the complex is based mostly on protein-protein interactions
2- TFIIA and TFIIB join after TFIID
3- RNA poly II joins with TFIIF
4- TFIIE and TFIIH (10 subunits) and TFIIJ enter the complex next
5- TFIIH initiates formation of open complex
6- CTD (carboxyl-terminal domain) of RNA Poly II is phosphorylated by TFIIH
7- factors are released (left bind) except TFIIH

Question 26

transition from initiation to elongation for RNA poly II transcription depends on…
explain

Answer 26

TFIIH- a very large multi-subunit protein with several enzymatic activities and functions
- helicase activity- responsible for initial melting of DNA at promoter to form OPEN complex
- kinase activity- phosphorylation of CTD (carboxyl terminal domain) of the RNA pol II
- helicase activity- two subunits (XPB & XPD) are involved in TCR (transcription-coupled repair)

Question 27

describe CDT phosphorylation by TFIIH

Answer 27

CTD: multiple repeats of the 7 AA sequence
- CTD during initiation is not phosphorylated
- TFIIH phosphorylates every serine 2 and serine 5 within CTD
- RNA poly II loses affinity for GTF’s and clears promoter
- beginning of elongation is the act of promoter clearance
- phosphorylation state of CTD determines what protein/protein complexes interact with RNA poly II
- elongation, transcript modification, and transcript cleavage are all dependent on modification state of CTD

Question 28

overview of transcription at RNA poly II promoters

Answer 28

1- Poly II recruited to DNA by TF’s
2- formation of transcription bubble
3- phosphorylation of CTD during initiation
4- elongation
5- dephosphorylation of CTD; transcription terminates

Question 29

transcription initiation for RNA poly II in vivo requires…

Answer 29

mediator complex at promoters
mediator: between specific TF’s bound at upstream promoter elements and enhancers AND the RNA pol II complex and general TF bound at core promoter

*mediator complex helps to integrate TF’s and RNA pol II with signals (activators and repressors) very far from the promoter

Question 30

describe termination of transcription of RNA poly II

Answer 30

Pol II termination does not occur at a conserved site or distance from the 3’ end of mature RNA’s
- mammals: takes place anywhere from a few base pairs to several kb pairs downstream from 3’ end of mature transcript
- polyadenylation signal sequence (typically AAUAAA) is present in the primary transcript (and directly encoded by DNA)
- factors responsible for cleavage of primary transcript bind to the AAUAAA sequence, resulting in cleavage somewhat downstream from that position

Question 31

in eukaryotes, the RNA poly II CTD must be phosphorylated before…

Answer 31

transcription can proceed from initiation to elongation