12: DNA to RNA (Transcription) Flashcards
(25 cards)
Transcription
1+2
DNA -> RNA
conserved across organisms
Complete process with multiple critical steps
DNA replication
summary
2
- separate 2 strands of DNA
- generate new daughter strand for each parent template
Replication fork?
2 strands
Leading strand: 3’5 strand is 5’-3’ synthesis
Lagging strand: 5’3 strand synthesised in okazaki fragments
Replication fork? whats involved
dna helicase - unwinding DFNA
sliding clamp and clamp loader
DNA primase - synthesis of new daughter DNA strand
Single strand DNA binding protein - protect/cover untranscripted parts of lagging strand
DNA Primase
how does it work + req?
match template strand and form phosphoester bond with new nucleotide incorporation
> req. correct positioning of incoming strand !!
DNA Fidelity
2 parts + error rates ig
DNA polymerase error rate: 1:10^5
> only possible in 5’3’ direction
Proof reading: 3’5’ exonuclease
> reduce error rate 1:10^2
Polymerases in E.coli
Pol III responsible for replication !!
Pol I = okazaki frag. processing
Pol II = lagging strand synthesis
Mammalian Polymerases
Many complex polymerases
with same basic mech but diff added functionality !!! `
DNA polymerase Structure
3 parts
Exonuclease region - for proofreading
Palm + Thumb + Finger regions: provide IA
+ Cation in active site
editing (proofreading) form and polymerase form !! (2 diff regions)
DNA polymerase active site?
3 steps
+ correct base? + incorrect base?
Enzyme starts in open position
upon base pairing with CORRECT dNTP
finger region undergoes conformation change/rotates
= closes/completes the active site
incorrect base pair = enzyme stays open
How is misincorporation error rate in DNA polymerase so low?
conf change w/ substrate coupling reduces error of misincorporation !
DNA Polymerase Mechanism
its metal ion dependant !!
- Incoming NT
B-site ion stabilised with this IA - Binding of Mg to A site (now there is ion in both A and B site)
= correct alignment of incoming NT
= water assisted deprotonation - 3rd metal ion at C
replaces side chain and inc stability of product
DNA polymerase Fidelity
1+3
1+3
Selectivity against misincorporation:
> shape complimentary between added bp and active centre
> active centre closed after direct match (stays open for mismatch)
> selection for dNTPS by steric gate !
Proofreading after incorporation:
> extension slows down after mismatch
> 3’5’ exonuclease site different from polymerase active centre
> mismatched end transferred over there
DNA Polymerase summary
DNA polymerase has diff domains
+ correct dNTP = finger region moves inward to close active site and properly position it
+ incorrect dNTP = conf change, transferred to second nuclease site
error rate: 1:10^8
Ribose vs deoxyribose
Uracil vs Thymine
OH -> H
(the one in the middle of all 3 OH)
CH -> C-CH3
on left of amide bond `
RNA polymerase cycle/types for bacteria
ONLY single type of RNA polymerase
> synth. all RNA
- polymerase complex formed at promotor
- unwinds DNA
- abortive initiation (short, ineff. unproductive)
- promoter clearance and sigma factor removal
- elongation !! (v productive)
- termination at hairpin formation
> destab polymerase hold on RNA
promoter seq: TATA region upstream~
RNA polymerase in eukaryotes
RNA Poly I: rRNA
RNA Poly II: ALL PROTEIN ENCODING GENES (regular RNA)
RNA Poly III: tRNA
Eukaryotic transcription by Poly II
Req many transcription factors and recruitment
> recognition of TATA: TFIID
> stabilisation
> unwinding DNA : TFIIH
> release RNA from promoter: TFIIH
TATA box 25 bp upstream of transcription start
Eukaryotic transcription + activator proteins?
can attach to enhancers (binding site for activators)
can be 1000s of bases away from TC start
> uses mediator that is very long and binds to both TC start and activator !! (coordination)
RNA Polymerase II Structure
Eukaryotes
Promo rec. loop: recognition of promoter region
N-term domain: DNA binding
C-term domain: not involved in core function
> some homologs to DNA polymerase
BUT NO EXONUCLEASE DOMAIN !!
RNA Polymerase II structure across organisms
2
Complex core function is conserved across species
> added additional areas for regulation prob
RNA Polymerase Mech
Active site related to DNA poly:
Catalyses formation of phosphoester bond between alpha-P of Nucleoside to OH-nucleotide
3 -‘ive residues coordinate 2Mg+ for structure allignment
Nuc attack onto inline alpha-phosphate
> req H2O
RNA Polymerase structural changes during mech?
Conf change results in active site closure after correct base pair formed !
> same as DNA polymerase
> but diff region changes ofc
Struc change of TRIGGER LOOP - RNA polymerase
(DNA poly = Finger loop)
Fidelity of RNA polymerase
- Selectivity against misincorporation
> shape complimentary of watson crick-bp and active centre
> active centre closes upon match
> selection for rNTPS by H-bond formation to Asn !! - Proofreading after incorporation
> extention slows
> RNA BACKTRACKING !!
> Active centre is tuneable: switches from polymerase to nuclease mode
