CMB2001/L01 Transcription Flashcards
Describe the central dogma.
DNA -> RNA -> Protein
Define transcription.
Transfer of genetic information from dsDNA to ssRNA
Briefly describe transcription in prokaryotes. (3)
RNA polymerase binds to promoter region with sigma70
Forms open complex and moves along DNA in transcription bubble
Sigma70 and RNA pol released
Define promoters.
Cis acting DNA regulatory element through which transcription is initiated and controlled
Where are promoters located?
Within 60bp of transcription start site
What defines the efficiency of transcription?
Similarity of promoter to consensus sequence
Give 3 eukaryotic promoters.
TATA box
Initiator (Inr)
MTE (motif tan element)
DPE (downstream core promoter element)
BRE (TFIIB recognition element)
Describe the TATA box. (3)
Eukaryotic promoter element
Located -31 to -26
Pyramidine-rich sequence
TATAA/TAA/TA/G
Describe the initiator (Inr).
Eukaryotic promoter element
-2 to +4
YYANA/TYYY
Describe the MTE (motif tan element). (3)
Eukaryotic promoter element
+18 to 28
CG/CAA/GCG/CG/CAAC
Describe the DPE (downstream core promoter region). (3)
Eukaryotic promoter element
+28 to 32
A/GGA/TCGTG
Describe the BRE (TFIIB recognition element). (3)
Eukaryotic promoter element
-37 to -32
G/CG/CG/AGGCC
How are eukaryotic promoters different from prokaryotic?
Eukaryotic promoters are looser and more flexible
Not all elements will be within one promoter
How can CpG islands affect gene transription?
Methylation is associated with silencing or switching off transcription
Which residues are often methylated in mammals?
C residues followed by G
Which residues escape methylation in mammals (hypomethylation)?
C residues in CpG islands
What are UAS/enhancers and URS/silencers?
Activator binding sites
Repressor binding sites
Describe reporter analysis.
Reporter genes encode proteins whose levels can be measured easily
Amount of reporter protein provides measure of gene expression
What can reporters be used to identify? (3)
When/where a gene is expressed
What signals it responds to
What factors and sequences control its expression
What is the target gene and location of RNA pol I?
rRNA (28S, 18S, 5.8S)
Nucleolus
What is the target gene and location of RNA pol II?
mRNA, snRNAs, miRNAs
Nucleus
What is the target gene and location of RNA pol III?
tRNA, 5S RNAs, U6 RNA, 7S RNA
Nucleus
Compare the structure of prokaryotic and eukaryotic RNA polymerase.
Similar structure but eukaryotic enzyme has more subunits
Bacterial - B, B’, a x2, w subunits
Eukaryotic pol II - 12 subunits
Give the 6 general transcription factors (GTFs) in eukaryotes.
TFIIA
TFIIB
TFIID
TFIIE
TFIIF
TFIIH
Give 3 features of eukaryotic GTFs.
RNA pol specific
Multi component factors
Form a complex on TATA box
Recruit RNA pol II to promoter
Direct initiation at start-site
Give the order of pre-initiation complex assembly.
IID
IIA
IIB
IIF & RNA pol II
IIH & IIE
Describe transcription initiation by RNA pol II. (5)
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
What is the function of TFIID?
Binds to TATA box (core promoter)
Recruits TFIIB
13 subunits
What is the function of TFIIA?
Stabilises TFIID binding
Anti-repression function
3 subunits
What is the function of TFIIB?
Recruits RNA pol II - TFIIF
Important for start site selection
1 subunit
What is the function of TFIIF?
Assists TFIIB recruit RNA pol II
Stimulates RNA pol II elongation
2 subunits
What is the function of TFIIE?
Helps recruit TFIIH and modulates TFIIH activity
2 subunits
What is the function of TFIIH?
Promoter melting and clearance
CTD kinase activity
DNA repair coupling
9 subunits
What 2 parts can TFIIH be divided into?
CORE
CAK
What does the CAK module of TFIIH contain?
A kinase that phosphorylates the CTD of RNA pol II
What ATPase does TFIIH contain and what does it do?
XPB (or Ssl2)
Key role in promoter melting
How does Ssl2(XPB) helicase form the open complex?
Uses energy from ATP hydrolysis
Pushes DNA into cleft where RNA polymerisation is catalysed
Creates torsional stress to form transcription bubble
Describe the structure of TFIID.
Central RNA pol II transcription factor
TATA binding protein + TBP associated factors (TAFs)
Describe TBP (TATA binding protein). (3)
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
What can TAFs do?
Promote interaction of TFIID with basal promoter elements
Interact with activators to promote transcription initiation
