Novel mutants of 23S RNA: characterization of functional properties

Single point mutations corresponding to the positions G2505 and G2583 have been constructed in the gene encoding E.coli 23S rRNA. These mutations were linked to the second mutation A1067 to T, known to confer resistance to thiostrepton (1). Mutant ribosomes were analyzed in vitro for their ability to direct poly(U) dependent translation, their missence error frequency and in addition their sensitivity to peptidyltransferase inhibitors. It was evident that the mutated ribosomes had an altered dependence on [Mg2+] and an increased sensitivity to chloramphenicol during poly(U) directed poly(Phe) synthesis. In a transpeptidation assay mutated ribosomes were as sensitive to chloramphenicol as wild-type ribosomes. However, the mutant ribosomes exhibited an increased sensitivity to lincomycin. An increase in translational accuracy was attributed to the mutations at the position 2583: accuracy increased in the order G less than A less than U less than C.