Transcription 1 Flashcards
Corden Lecture 1
What’s defines a transcription unit?
Promoter, Genes, Terminator
T/F. Bacteria couple transcription to translation
true
T/F. Eukaryotes couple transcription to translation
False, couple transcription to processing
4 Eukaryotic RNA Polymerase Families and Transcription Targets
1 - rRNA
2 - mRNA, small regulatory RNAs
3 - tRNA, 5S RNA, snRNA
RNA Polymerase Characteristics (3)
5’ -> 3’ chain elongation
Conserved a.a. between species are in catalytic active site
Multi-subunit
Function: Sigma Factor (3)
- Destabalize nonspecific binding to non-promoter DNA
- Stabilize specific binding to promoter DNA
- Accelerate search for promoter DNA
Structure: Sigma Factor Regions and Functions (4)
Region 1.1 - binds RNA pol where template strand would be
Region 2 - binds -10 of promoter
Region 3.2 - binds RNA pol, important for abortive initiation
Region 4 - binds -35 of promoter
Transcription Initiation Steps (3)
Promoter Binding, Promoter Unwinding, Promoter Clearance
Mechanism of Promoter Melting in Bacteria
1) Sigma factor binds promoter
2) Isomerizes A in -10 region
3) Twist in downstream region leads to relief of negative supercoil, allow ATP independent melting
4) DNA replaces Region 1.1 of sigma factor
5) Open complex is formed
Mechanism of Abortive Transcription in Bacteria
1) Open complex is formed
2) Oligonucleotides are formed in active site
3) With enough [NTP], oligonucleotide will displace region 3.2 of sigma factor
4) Get promoter clearance
Two types of promoters in Eukaryotes
Sharp type promoters: Have TATA box and specific initiator site
Borad type promoters: nonspecific transcription start site, usually consists of CpG islands
RNA Pol II Promoter Elements (3)
TATA box, Inr, BRE (B recognition element), DPE (downstream promoter element)
Optional: MTE, DCE, XCPE1
Function: TFIIA
Stabilize binding of TBP and TFIIB
Function: TFIIB
Promoter recognition, stabilize early transcribing complex, binds BRE
Function: TFIID
Consists of TBP, TAFs,
involved in promoter recognition, DNA bending to interact w/ regulatory factors
binds TATA, INR, DPE
Function: TFIIE
Recruit TFIIH
Function: TFIIF
capture non-template strand upon melting
optional: suppress non-specific DNA binding
Function: TFIIH
unwind promoter DNA with 3’->5’ ATP dependent helicase
Phosphorylate CTD
What happens if you mutate TFIIB?
Get altered start site selection
What is the Pol II-CTD?
Repeating heptapeptide that can be modified. Different residues are modified depending on state of transcription
Function: DSIF
Binds to NELF to initiate promoter proximal pausing
Function: NELF
Locks Pol II in inactive conformation by locking DNA-RNA hybrid
Function: P-TEFb
inactivates DSIF and NELF through phosphorylation, allows Pol II release
Why does Pol II pause at promoters? (2)
1) Ensure that 5’ end of mRNA is capped.
2) Adds layer of regulation, especially for developmental genes or genes involved in stimuli
How does RNA Pol translocate?
Thermal ratchet mechanism. Brownian motions allows Pol to move, but NTP binding + hydrolysis traps it in forward translocated state
What kind of supercoils appear in front and behind Pol and what enzymes deal with them?
Behind: Negative supercoils, Topoisomerase In front (bacteria): Positive supercoils, gyrase
What are the two types of pause sites in elongation?
Class 1 (bacteria) is related to rho-independent terminators.
Class 2: weak hybrids (rU:dA hybrid) cause Pol backtrack
Mechanism: Backtracking
1) Stalled polymerase backtracks
2) RNA goes through secondary channel
3) GreB (Bacteria) / TFIIS (Eukaryotes) cleaves RNA
uses Mg2+ ions to facilitate cleavage
Mechanism: Rho Independent Termination
1) GC rich dyad sequence is transcribed
2) Dyad sequence forms hairpin and causes pausing
3) rU:dA hybrid downstream is unstable, causes release
Mechanism: Rho-Dependent Termination
1) C-rich rut site is transcribed
2) Rho binds the site, uses ATP hydrolysis to move along RNA
3) crashes into Pol and destabilizes complex
What protein regulates Rho?
NusG
Negatively regulates during normal elongation
Positively regulates when there is stress (ribosome stalls)
Eukaryotic Termination Pathways (2)
Allosteric model and Torpedo Model
