Macromolecular Machines - Summary of Publications Biochemistry 4000 Dr. Ute Kothe

Kinetic Mechanism of A-Site Binding Proofreading Initial Selection

Accuracy in protein synthesis Proofreading Initial Selection 1. 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 n 2.Catalytic site: 3’hydroxyl of primer strand, 2 catalytic Mg2+ ion 3.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-Polymerase Mismatch-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