Accuracy in protein synthesis
Proofreading
Initial Selection1. Kinetic proofreading:
Two selection phases
separated by irreversible
step (GTP hydrolysis =
energy investment)
Multiplies accuracy
2. Induced Fit:
Forward steps significantly
Faster for correct
substrate,Increases speed
and accuracy
3. Stability difference
Back steps faster for
incorrect substrate
RNA-Polymerase
• can use RNA template
RdRP activity (RNA-dependent RNA Polymerase activity)
• Assay to check for elongation of RNA:
• fluorescently labeled RNA
• addition of selected NTPs which are complementary to template
• analysis of fluorescent, elongated RNA product on high-percentage gel (single nucleotide resolution)
RNA-Polymerase• RNA(product)-RNA(template) duplex is an A-type helix
• usual RNA(product)-DNA(template) duplex is A-type helix as well
Both fit into RNA-Pol exit cleft
Significance:
• Evolution: RNA-Polymerase might have evolved shortly after RNA world to replicate RNA which still contained the genetic information
• Hepatitis Delta Virus: RNA-Pol + TFIIS might cleave RNA genome to allow for direct replication by RNA-Pol. (circumventing the need for reverse transcription to generate DNA)
DNA-Polymerase
1. Insertion site: incoming nucleotide pairs with template base n2. Catalytic site: 3’hydroxyl of primer strand, 2 catalytic Mg2+ ion3. Pre-insertion site: template base N prior to incorporation 4. Post-insertion site: grwoing end of duplex DNA (new base-pair n-
1 after translocation)5. DNA duplex binding region: binding of 4 base-pair (n-2, ..., n-5)
duplex to polymerase
DNA-PolymeraseMismatch-Induced Disruptions:- depending on type of mismatch
1. Disruption of template strand and pre-insertion site ***
2. Disruption of primer strand and assembly of catalytic site
3. Disruption of template and primer strands
4. Fraying of DNA at insertion site
*** G-T mismatch in postinsertion site AND G-T mismatches in position n-2 and n-3
“Short term memory”: disruption of active site for at least 3 rounds of elongation
1.
4.
3.
2.