BMED5207 unit 2 - slides

Why does transcriptional control matter?

cell circumstances change, cell identities change over time, many different cell "types" in an organism

How can the cell regulate transcription?

transcriptional control regions, proteins that bind to DNA to alter transcription, manipulation of chromatin structure -access to genes

RNA polymerases in eukaryotes

RNA polymerase I, II, III

general transcription factors (GTFs) are essential for:

recognition of the core promoter, assembly of preinitiation complex

Eukaryotic RNAs

- are capped

- are tailed

- have their introns removed.

- mono-cistronic

RNA polymerase 1 -RNA transcribed

rRNAs (pre-rRNA)

RNA polymerase II - RNA transcribed

mRNAs (and snRNAs, siRNAs, miRNAs)

RNA polymerase III - RNA trnascribed

tRNAs (and 5s rRNA, snRNA U6, 7s RNA, other small stable RNAs)

RNA polymerase components

RPB1 and 2 (RNA Polymerase B1, RNA Polymerase B2)

Prokaryote polymerase nomeclature

greek letters

Eukaryote polymerase nomenclature

arabic numerals and english letters

Which eukaryote polymerases are most similar?

Pol I and III

What makes Pol II unique (compared to the other polymerases)?

unique alpha-like-subunits and unique CTB on RPB1

What is CTD?

C-terminal domain

What are unique to each polymerase?

additional subunits (enzyme specific)

How many additional subunits does pol I have?

5

How many additional subunits does pol II have?

3

How many additional subunits does pol III have?

7

Are all eukaryotic polymerase subunits needed for transcription?

yes

Clamp Domain

clamp domain moves to hold DNA template and transcript; clamp domain on RPB1, wall domain on RPB2

DSIF

elongation factor; supports by stabilizing the closed clamp

C-terminal domain (CTD)

unstructured C-terminal region of RPB1 with heptapeptide repeats

Consensus repeat/sequence of CTD

YSPTSPS

How many consensus repeats do yeast have on CTD?

26

How many consensus repeats do vertebrates have on CTD?

52

Are the repeats on CTD essential?

yes, <10 repeats is lethal

CTD phosphorylation

CTD is initially unmodified, after transcription initiation, CTD is phosphorylated, Phospho-CTD supports other binding regulatory proteins

What does CTD stand for?

C-terminal Domain

Where is CTD phosphorylated?

mainly serines 2 and 5 of repeats

What does Phospho-CTD support?

mRNA processing and Chromatin remodeling

Transcriptional Control Region

a DNA sequence that a protein binds to, DNA-protein interaction influences transcription

Example of transcriptional control region

promoters

What does TSS stand for?

transcriptional start site

What determines a transcriptional start site?

focused/regulated promoters & dispersed/housekeeping promoters

Focused/regulated promoters

TATA boxes, initiator sequences, BREs, DPE

What does BREs stand for?

TFIIB Recognition Element

What does DPE stand for?

Downstream Promoter Element

What are dispersed/housekeeping promoters?

CpG islands

What are CpG islands?

region of DNA that is rich in sequences that go CGCGCG, p stands for phosphodiester bond between them

Do all mRNAs start at the same site for focused/regulated promoters?

yes

Do all mRNAs start at the same site for dispersed/housekeeping promoters?

no

What is the expression level of genes that focused/regulated promoters are found in?

varied expression levels

What do focused/regulated promoters do?

help position RNA polymerase on start site, binding sites for general transcription factors

Where are BRE found?

~ -37 to -32

Where is the TATA box found?

~ -31 to -26

Where is Inr found?

-2 to +4

Where is DPE found?

+28 to +32

What does DPE stand for?

downstream promoter element

What is the peach box (-37 to -32)?

BRE

What is the orange box (-31 to -26)?

TATA box

What is the yellow box (-2 to +4)?

Inr

What is the blue box (+28 to +32)?

DPE

What is the expression level of genes that dispersed/housekeeping promoters are found in?

constant expression levels

Do dispersed/housekeeping promoters directly position RNA Pol?

no

Which types of promoters (Dispersed/housekeeping vs focused/regulated) have multiple TSS?

dispersed/housekeeping

Why are CpG islands nucleosome free regions?

they are less flexible and hard to bend around nucleosomes

CpG repeats in mammalian DNA

normally pretty low due to methylation (5-methyl C)

What can 5-methyl C spontaneously deaminate to?

T (C^meT → TG)

What can C spontaneously deaminate to?

U; DNA repair enzmes will convert back to CG; CG → UG → CG

What is methylation of CpGs a signal for?

Transcriptional repression

Transcription sequence in eukaryotes

Recruitment, pre-initiation complex, initiation, pausing, elongation, termination

What does GTF stand for?

General Transcription Factors

What are GTFs?

proteins or multi-subunit protein complexes

What are the different GTFs?

TFIIA, B, D, E, F, H

Functions of GTFs

position RNA Pol II at TSS, aid in binding to promoter core elements, help separate DNA strands to allow template strand to enter Pol active site

Are GTFs required for all genes transcribed by RNA Pol II?

yes

Are GTFs highly conserved across all eukaryotes?

yes

TFIIA subunits

3

TFIIA function

stabilize TBP-TATA

TFIIB subunits

1

TFIIB function

stabilize TFIID-TATA, start site selection

TFIID subunits

TBP + ~ 13 TAFs

TFIID function

core promoter recognition

TFIIE subunits

2

TFIIE function

TFIIH recruitment

TFIIF subunits

2

TFIIF function

chaperone Pol II to promoter

TFIIH subunits

10

TFIIH function

CTD kinase, helicase, DNA repair

First step in the assembly of the pre-initiation complex (TATA promoter example)

TATA promoters recognized by TFIID; TBD binds first (“saddle” that rides DNA)

Second step in the assembly of the pre-initiation complex (TATA promoter example)

TFIIA binds DNA and TBP to stabilize

Third step in the assembly of the pre-initiation complex (TATA promoter example)

TFIIB binds DNA and TBP, later separate strands

Fourth step in the assembly of the pre-initiation complex (TATA promoter example)

RNA Pol II and TFIIF bind the complex

Fifth step in the assembly of the pre-initiation complex (TATA promoter example)

TFIIE binds complex and forms binding site for TFIIH

Transcription initiation from the PIC step 1

TFIIH helicase subunit opens the DNA

What is the TFIIH helicase subunit?

XPB

PIC meaning

Pre-initiation complex

Transcription initiation from the PIC step 2

TFIIB and Pol II melt DNA, opening bubble (open complex)

Transcription initiation from the PIC step 3

RNA Pol II initiates transcription

Transcription initiation from the PIC step 4

TFIIH kinase domain phosphorylates RPB1 CTD (serine 5 of repeats) and an enzyme complex binds phospho-CTD and caps mRNA

Transcription initiation from the PIC step 5

GTFs dissociate from the promoter

What portion(s) of the promoter does TFIIB bind?

BRE

What portion(s) of the promoter does TFIID bind)

Inr and DCE

The CTD code

the presence of specific phosphorylation recruits proteins to regulate initiation, pausing and elongation

No phosphorylation (CTD code)

RNA Pol II assembled into PIC

Ser-5 phosphorylation (CTD code)

Transcriptional initiation and pausing (phosphorylated by TFIIH, recruits capping complex, recruits elongation inhibition factors (NELF/DSIF))

Ser-2/5 phosphorylation (CTD code)

processive elongation (phosphorylation by P-TEFb, recruits splicing complex)

RNA Pol II pausing and phosphorylation

in PIC, TFIIF and TFIIE cover binding sites for NELF/DSIF; once initiation starts, GTFs separate opening up site; NELF/DSIF bind within 100 bases, pausing transcription; P-TEFb relieves pausing

How does P-TEFb relieve pausing?

phosphorylating NELF and DSIF, phosphorylating serine 2 of CTD repeats, NELF dissociates, other elongations factors can bind

How is P-TEFb recruited to euchromatin?

by BRD4 (BRD4 has bromodomain which fins acetylated histones)