Peptidyl transferase activities of gapped-cp-reconstituted subunits containing 2′-deoxyribose modifications at A2451 using CC-puromycin as acceptor substrate

<p><b>Copyright information:</b></p><p>Taken from "Chemical engineering of the peptidyl transferase center reveals an important role of the 2′-hydroxyl group of A2451"</p><p>Nucleic Acids Research 2005;33(5):1618-1627.</p><p>Published online 14 Mar 2005</p><p>PMCID:PMC1065261.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p> () The reaction between -acetyl-Phe-tRNA and [P]CC-puromycin was carried out for 120 min, a time point that corresponded to the endpoint of the reaction catalyzed by reconstituted wild-type large subunits. The product -acetyl-Phe-CC-puromycin (CC-Pmn-AcPhe) was resolved from CC-puromycin (CC-Pmn) by gel electrophoresis (). The control reaction (ctrl) contained the whole reaction mixture except ribosomal particles. The relative yields of product formation by gapped-cp-reconstituted subunits containing adenosine (wt), 2′-deoxyadenosine (dA), or the deoxy-abasic analog (d-aba) at 2451 are shown below the gel. () The initial rates of peptide bond formation catalyzed by the wt or the deoxy-A2451-modified large ribosomal subunit were determined from experimental points within the first 45 min of incubation. During this incubation time, no product formation with ribosomal particles carrying the deoxy-abasic site analog at position 2451 could be measured (n.d.). The rates were normalized to the rate of reconstituted subunits containing the synthetic wild-type RNA fragment (wt)