The Regulation of Transcription in Eukaryotes

Question 1

What determines the identity and capability of a cell

Answer 1

relative abundance of gene products

Question 2

Describe transcription - the basics

Answer 2

• first step in converting DNA to gene products
• first step in genotype to phenotype
• primary regulatory gateway for gene expression

Question 3

Number of genes per cell:

Answer 3

40,000

Question 4

Number of transcripts per cell:

Answer 4

300,000

Question 5

Number of proteins per cell:

Answer 5

1,000,000,000

Question 6

Describe the amplification of transcripts in a cell

Answer 6

~5x

Question 7

What is the average transcript number per gene?

Answer 7

~10

Question 8

Describe the amplification of proteins in a cell

Answer 8

~130x
amplification

Question 9

What is the average protein number per gene?

Answer 9

~1,300

Question 10

Transcription in eukaryotes is regulated at…

Answer 10

three temporally distinct stages

Question 11

When is transcription in eukaryotes regulated?

Answer 11

• initiation
• elongation
• termination

Question 12

Describe transcription regulation at initation

Answer 12

• transcription factors
• promoters
• pre-initiation complex

Question 13

Describe transcription regulation at elongation

Answer 13

• TFIIS
• nucleosome/chromatin remodeling

Question 14

Describe transcription regulation at termination

Answer 14

Cleavage and polyadenylation

Question 15

Describe transcription factors

Answer 15

• the first step transcriptional regulation
• composed of one or more DNA binding domains and protein-protein interaction domains.
• bind to short DNA sequence elements (6-20bp)
• recruit (or block recruitment) of other proteins
• act as activators or repressors

Question 16

What the humans genes coding for transcription factors

Answer 16

• 1600 genes
• 8%

Question 17

Describe transcription factor gene families

Answer 17

• ~20
• ~50% of transcription factors in human genome are C2H2 zinc finger transcription factors

Question 18

Describe the mechanics of transcription factors

Answer 18

• DNA binding domain binds to short sequences in the nucleosome
• recruit enzymes that alter nucleosomes
• recruit the PIC
• GTFs

Question 19

PIC

Answer 19

pre-initiation complex

Question 20

General Transcription Factors

Answer 20

GTFs

Question 21

List the GTFs

Answer 21

• TFIIA
• TFIIB
• TFIID
• TFIIE
• TFIIF
• TFIIH

Question 22

Describe TFIID

Answer 22

• 13 subunit protein complex
• first recruited component of the pre-initiation complex
• binds to TBP

Question 23

TBP

Answer 23

TATA binding protein

Question 24

Describe the TFIID/TBP complex

Answer 24

• assembles without interacting with DNA
• TFIID/TBP complex binds DNA via TBP subunit.
• first stage of pre-initiation complex assembly.
• can be recruited without a TATA binding site for TBP

Question 25

Describe the TATA box

Answer 25

- consensus sequence is TATAWAW - W is either A or T

Question 26

Describe TFIIA

Answer 26

- 3 subunit protein complex - binds to TFIID/TBP - stabilises TBP interaction with DNA

Question 27

Describe TFIIB

Answer 27

- single protein - facilitates recruitment of RNA polymerase II

Question 28

Describe TFIIF

Answer 28

- TFIIF – tetramer - Pol II recruited at same time. - stabilises Pol II interaction with TFIIB and TFIID

Question 29

Describe Pol II

Answer 29

- RNA polymerase II - 12 subunit complex

Question 30

tetrameric

Answer 30

two subunit complex

Question 31

Describe TFIIE

Answer 31

- 2 subunit complex - recruits TFIIH

Question 32

Describe TFIIH

Answer 32

- 10 subunit complex - acts as a DNA helicase

Question 33

Describe the two main function of TFIIH

Answer 33

- opening the promoter to load the polymerase - phosphorylating Pol II CTD – promoter escape

Question 34

What happens on pre-initiation assembly?

Answer 34

- phosphorylated Pol II recruits capping enzyme - first nucleotide that exits the polymerase gets capped - cap protects the RNA from degradation - polymerase escapes the promoter and proceeds to transcribe the gene

Question 35

transcription factors act

Answer 35

in concert

Question 36

If one transcription factor is mutated

Answer 36

All efforts are lost

Question 37

The combinatorial action of transcription factors ...

Answer 37

specifies cell identity and function

Question 38

Describe elongating polymerases

Answer 38

- not under control of TFs or PIC

Question 39

Describe polymerase gene transcription

Answer 39

- average gene ~20,000 bp: 4.5 mins @ 72 nucleotides sec-1 - longest gene ~2.2 million bp: 8.5 hours @ 72 nucleotides sec-1

Question 40

What happens if transcription goes wrong?

Answer 40

- elongating polymerase backtracks along the DNA - 3’ OH no longer available for extension - backtracking cannot be recovered by polymerase - RNA must be cleaved to produce new 3’OH - once cleaved, polymerase can resume

Question 41

Describe cleavage on elongation backtracking?

Answer 41

- TFIIS - creates two domains

Question 42

Describe the average gene

Answer 42

- ~20,000 bp - ~90 nucleosomes

Question 43

Describe the longest gene

Answer 43

- ~2.2 million bp - 10,000 nucleosomes

Question 44

How does Pol-II navigate nucleosomes

Answer 44

with the help of a large cohort of chromatin remodeling enzymes

Question 45

Describe transcription termination

Answer 45

- PAS encoded in the DNA - Polymerase transcribes past the PAS - CAP complex binds Pol II and PAS in RNA grabs the polymerase and RNA

Question 46

PAS

Answer 46

polyadenylation signal - AAUAAA

Question 47

CAP

Answer 47

- Cleavage and polyadenylation complex - 83 proteins