Peptidyltransferase assay systems

Information on archaeal translation is essentially based on poly(U)- and poly(UG)-programmed cell-free systems and on peptidyltransferase assay systems. Poly(U)-directed systems have been used to monitor the reconstruction of archaeal ribosomal subunits and the susceptibility of archaea to protein synthesis inhibitors. 

Perhaps the most interesting conclusion from the peptidyltransferase assays is the evidence that ribosomes from extremely thermophilic archaea display significant peptidyltransferase activity (both uncoupled and coupled) at temperatures (37°C) well below that required for poly(U)-directed polypeptide synthesis. Therefore, in the thermophile systems high temperature appears to activate reactions other than peptide bond formation. 

It is usually accepted that the 16-membered-ring macrolides inhibit peptidyltransferase activity [79, 88, 89], because they inhibit puromycin reaction although poorly. The reaction is used as an assay system for peptidyltransferase activity, because puromycin characteristically interrupts peptide bond formation by virtue of its structural similarity to the 3 end of aminoacyl-tRNA. Puromycin enters the A site (the so-called aminoacyl site) on the ribosome and is incorporated into either a nascent polypeptide or into A-acylaminoacylate, consequently causing premature release of puromycinyl polypeptide or A-acylaminoacyl-puromycin from the ribosome. 

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