Control of Gene Expression Flashcards
What are the types of RNA polymerase found in prokaryotes and how are they different?
Core
Holo - contains sigma subunit
What are the core components of prokaryotic RNA polymerase?
Beta subunit
Beta prime subunit
Alpha subunits (2)
Which subunit of prokaryotic RNA polymerase binds to the promoter region?
Sigma
Which part of the prokaryotic RNA polymerase subunit helps to stabilise RNA polymerase on the RNA?
The alpha subunits
What are the three different ways that RNA polymerase can be stabilised on the RNA?
alpha subunits
- UP element binding
- CAP (catabolite activating protein) class 1
- CAP class 2
What is the UP element and how does it work?
UP element is located before -35 region (on 5’ end - before start of translation). It binds the the C-Terminal Domain (CTD) of the alpha subunits of RNA pol. to stabilise it on the RNA.
What is CAP and how do the different classes work?
For CAP - DNA binding domain at C-terminus
Stabilises the pol. on the promoter
- 60 is first CAP binding site (22bp of DNA) IN LAC Z
- 40 is first CAP binding site, GalR(repressor) at -60 IN galP1
CLASS 1 = only CTD
CLASS 2 = CTD and NTD of RNA pol.
CAP has two regions conforming to transactivation domain, one binds to alpha c-terminal domain of RNA pol and other binds to n-terminal domain of RNA po
What happens if different activating regions (AR) of CAP are altered?
AR1 - does affect formation of closed complex, reduces recruitment (RNA pol. binding), but does not affect closed>open complex rate
AR2 - affects opening up of RNA for transcription (isomerisation rate) - closed>open complex (melting of DNA), formation of closed complex not affected (DNA-RNA pol. binding)
What are AR1 and AR2 and what do they do?
AR = activation region in class 2 CAP binding , RNA pol. binds to AR1 and AR2.
AR1 = forming closed complex between RNA and RNA pol. (stabilising)
AR2 = opening up RNA pol. from closed complex for transcription
PROKARYOTES
Components (basic) of a prokaryotic gene
CAP site/UP site Promoter Operator Structural Gene Transcription Factors
What is an operon?
Genes of similar function ordered into groups, controlled by single promoter.
WHY?: Because they are likely in response to a particular condition where they would all be needed
Types of Histone Modification
Phosphorylation
Acetylation - neutralises charge (tails are +ve charge/acetyl groups -ve charge)
Methylation
Ubiquitination
Promoter region in Eukaryotes
-30 TATA box
Promoter Region in Prokaryotes
-35 and -10 (TATA equivalent) sites
What is the intermediate region between the promoter regions in prokaryotes?
Important - 15-21 BP long
May alter how RNA pol. binds to RNA
WHY? 10 BP per turn of alpha helix, length of region -> whether both promoter regions are on the same side for RNA pol. binding
How does bacteria have differential gene expression?
Change of sigma factor decides promoter that RNA pol. binds to.
How? Slightly different sequence for promoter binding area.
IMPORTANCE OF SIGMA FACTOR (EVIDENCE):
- CROSS-LINKING
MIX RNA + PROMOTER SEQ. +UV LIGHT
SEE WHERE STICKS
CROSS-LINKS BETWEEN SIGMA AND DNA - SUPPRESSOR OR ‘SECOND-SITE’ MUTATIONS
MUTATE TO AFFECT THE ABILITY OF RNA POL. TO BIND TO PROMOTER
SELECTING FOR RNA POL. THAT CAN RECOGNISE PROMOTER
FIND OUT WHERE SUPPRESSOR MUTATIONS ARE (WHERE DOES NOT WORK)
SIGMA FACTOR - DELETING THE N-TERMINUS OF SIGMA FACTOR ALLOWS DNA BINDING
PART OF SIGMA FACTOR INHIBITS BINDING (N-TERMINUS)
REMOVE N-TERMINUS -> EASIER BINDING
TBP
Tata binding protein (TO TATA BOX)
How is TBP unusual?
Does not bind using alpha helices
Binds using beta-sheet secondary structure in minor groove of DNA (in other cases - with alpha would by major groove)
-> Distortion, bending and binding of DNA (-> melting)
THE STEPS - PRE-INITIATION COMPLEX
Eukaryotes
- TBP binds
- TB2F binds
- TF2E binds
- TF2H binds
Eurkaryotes
TF2H role in the initiation complex
TFIIH (2H) - kinase CDK7 is a subunit, which phosphorylates C-terminal domain of RNA pol. II at serine 5 in transition from initiation complex to elongation complex
Eukaryotes
ELONGATION COMPLEX FORMATION
Eukaryotes
CTD very IMPORTANT
Synthesis mRNA - transcription factor dis-assemble, so RNA pol. Can transcribe
Phosphorylate large RNA pol. II subunit (RPB1 - large subunit)
What does CDK stand for in CDK9?
Cyclin-dependent kinase
How are CDKs involved in the Elongation Complex formation?
CDK9 & CDK12
Phosphorylate at serine position 2 of C-terminal domain (CTD of the RNA pol. II large subunit)