CMB2001/L01 Transcription

Question 1

Describe the central dogma.

Answer 1

DNA -> RNA -> Protein

Question 2

Define transcription.

Answer 2

Transfer of genetic information from dsDNA to ssRNA

Question 3

Briefly describe transcription in prokaryotes. (3)

Answer 3

RNA polymerase binds to promoter region with sigma70
Forms open complex and moves along DNA in transcription bubble
Sigma70 and RNA pol released

Question 4

Define promoters.

Answer 4

Cis acting DNA regulatory element through which transcription is initiated and controlled

Question 5

Where are promoters located?

Answer 5

Within 60bp of transcription start site

Question 6

What defines the efficiency of transcription?

Answer 6

Similarity of promoter to consensus sequence

Question 7

Give 3 eukaryotic promoters.

Answer 7

TATA box
Initiator (Inr)
MTE (motif tan element)
DPE (downstream core promoter element)
BRE (TFIIB recognition element)

Question 8

Describe the TATA box. (3)

Answer 8

Eukaryotic promoter element
Located -31 to -26
Pyramidine-rich sequence
TATAA/TAA/TA/G

Question 9

Describe the initiator (Inr).

Answer 9

Eukaryotic promoter element
-2 to +4
YYANA/TYYY

Question 10

Describe the MTE (motif tan element). (3)

Answer 10

Eukaryotic promoter element
+18 to 28
CG/CAA/GCG/CG/CAAC

Question 11

Describe the DPE (downstream core promoter region). (3)

Answer 11

Eukaryotic promoter element
+28 to 32
A/GGA/TCGTG

Question 12

Describe the BRE (TFIIB recognition element). (3)

Answer 12

Eukaryotic promoter element
-37 to -32
G/CG/CG/AGGCC

Question 13

How are eukaryotic promoters different from prokaryotic?

Answer 13

Eukaryotic promoters are looser and more flexible
Not all elements will be within one promoter

Question 14

How can CpG islands affect gene transription?

Answer 14

Methylation is associated with silencing or switching off transcription

Question 15

Which residues are often methylated in mammals?

Answer 15

C residues followed by G

Question 16

Which residues escape methylation in mammals (hypomethylation)?

Answer 16

C residues in CpG islands

Question 17

What are UAS/enhancers and URS/silencers?

Answer 17

Activator binding sites
Repressor binding sites

Question 18

Describe reporter analysis.

Answer 18

Reporter genes encode proteins whose levels can be measured easily
Amount of reporter protein provides measure of gene expression

Question 19

What can reporters be used to identify? (3)

Answer 19

When/where a gene is expressed
What signals it responds to
What factors and sequences control its expression

Question 20

What is the target gene and location of RNA pol I?

Answer 20

rRNA (28S, 18S, 5.8S)
Nucleolus

Question 21

What is the target gene and location of RNA pol II?

Answer 21

mRNA, snRNAs, miRNAs
Nucleus

Question 22

What is the target gene and location of RNA pol III?

Answer 22

tRNA, 5S RNAs, U6 RNA, 7S RNA
Nucleus

Question 23

Compare the structure of prokaryotic and eukaryotic RNA polymerase.

Answer 23

Similar structure but eukaryotic enzyme has more subunits
Bacterial - B, B’, a x2, w subunits
Eukaryotic pol II - 12 subunits

Question 24

Give the 6 general transcription factors (GTFs) in eukaryotes.

Answer 24

TFIIA
TFIIB
TFIID
TFIIE
TFIIF
TFIIH

Question 25

Give 3 features of eukaryotic GTFs.

Answer 25

RNA pol specific Multi component factors Form a complex on TATA box Recruit RNA pol II to promoter Direct initiation at start-site

Question 26

Give the order of pre-initiation complex assembly.

Answer 26

IID IIA IIB IIF & RNA pol II IIH & IIE

Question 27

Describe transcription initiation by RNA pol II. (5)

Answer 27

Helicase activity of TFIIH separates template strand at start-site (ATP using) Transcription starts (promoter clearance) and pol II is extensively phosphorylated on C-terminus domain TFIID & TFIIA may stay behind TFIIB, TFIIE, TFIIH released TFIIF moves down template with pol II

Question 28

What is the function of TFIID?

Answer 28

Binds to TATA box (core promoter) Recruits TFIIB 13 subunits

Question 29

What is the function of TFIIA?

Answer 29

Stabilises TFIID binding Anti-repression function 3 subunits

Question 30

What is the function of TFIIB?

Answer 30

Recruits RNA pol II - TFIIF Important for start site selection 1 subunit

Question 31

What is the function of TFIIF?

Answer 31

Assists TFIIB recruit RNA pol II Stimulates RNA pol II elongation 2 subunits

Question 32

What is the function of TFIIE?

Answer 32

Helps recruit TFIIH and modulates TFIIH activity 2 subunits

Question 33

What is the function of TFIIH?

Answer 33

Promoter melting and clearance CTD kinase activity DNA repair coupling 9 subunits

Question 34

What 2 parts can TFIIH be divided into?

Answer 34

CORE CAK

Question 35

What does the CAK module of TFIIH contain?

Answer 35

A kinase that phosphorylates the CTD of RNA pol II

Question 36

What ATPase does TFIIH contain and what does it do?

Answer 36

XPB (or Ssl2) Key role in promoter melting

Question 37

How does Ssl2(XPB) helicase form the open complex?

Answer 37

Uses energy from ATP hydrolysis Pushes DNA into cleft where RNA polymerisation is catalysed Creates torsional stress to form transcription bubble

Question 38

Describe the structure of TFIID.

Answer 38

Central RNA pol II transcription factor TATA binding protein + TBP associated factors (TAFs)

Question 39

Describe TBP (TATA binding protein). (3)

Answer 39

Can direct assembly of PIC on TATA-containing promoter in vitro Alone can NOT direct PIC assembly on TATA-less promoter Can NOT support activated transcription

Question 40

What can TAFs do?

Answer 40

Promote interaction of TFIID with basal promoter elements Interact with activators to promote transcription initiation