FUNCTIONAL-EFFECTS OF A G-BASE TO U-BASE CHANGE AT POSITION-530 IN A HIGHLY CONSERVED LOOP OF ESCHERICHIA-COLI 16S RNA

Any base change at position 530 introduced into Escherichia coli on a multicopy plasmid leads to cell death [Powers & Noller (1990) Proc. Na tl. Acad. Sci. U.S.A. 87, 1042-1046]. It was suggested that these muta nts cannot carry out chain elongation. To define more precisely the fu nction of base 530, we have studied ribosomes in which G530 was mutate d to U530. In vivo, U530 16S rRNA was incorporated into 30S subunits a nd could combine with 50S to make 70S ribosomes. 16S rRNA in vitro tra nscripts containing U530 were assembled into 30S ribosomes, and their activity was tested in defined steps of protein synthesis. Mutant 30S ribosomes were as active as wild-type in poly(U)-dependent poly(Phe) s ynthesis, P- and A-site tRNA binding, and 30S initiation complex forma tion. 30S initiation complexes, in the presence of 50S, could react wi th puromycin like the wild-type. The rate, extent, and position of cro ss-linking of AcVal-tRNA in the P site to 16S RNA were identical in mu tant and wild-type ribosomes. Although there appeared to be no defect in 70S initiation complex formation or in direct A-site binding of Phe -tRNA dependent on poly(U), U530 30S ribosomes were nevertheless defec tive in carrying out synthesis of fMet-Val dipeptide using natural mRN A. Mutant 30S ribosomes were also refractory to streptomycin-induced m isreading although no misreading was observed in its absence. The defe ct in U530 ribosomes may be due to a block in correct binding of the f irst aminoacyl-tRNA, to infidelity in translation due to frameshifting , or to some other aspect of A-site interaction related to the use of natural mRNA instead of poly(U).