LOCALIZATION AND CHARACTERIZATION OF THE GENE ENCODING RELEASE FACTORRF3 IN ESCHERICHIA-COLI

Two protein release factors (RFs) showing codon specificity, RF1 and R F2, are known to be required for polypeptide chain termination in Esch erichia coli. A third protein component has also been described that s timulates termination in vitro, but it has remained uncertain whether this protein, RF3, participates in termination in vivo or is essential to cell growth. We report (i) the purification and N-terminal sequenc ing of RF3; (ii) the isolation of transposon insertion mutants similar to miaD, a suppressor of a leaky UAA mutation affecting the gene miaA , leading to enhanced nonsense suppression; (iii) the localization of the affected gene on the physical map of the chromosome; and (iv) the cloning and sequencing of the wild-type gene, providing proof that it encodes the factor RF3. We designate the gene prfC. Two transposon ins ertions were shown to interrupt the coding sequence of prfC, at codons 287 and 426. The enhanced nonsense suppression in the insertion mutan ts shows that the product participates in termination in vivo. The iso lation of such mutants strongly suggests that the gene product is not essential to cell viability, though cell growth is affected. RF3 is a protein with a molecular weight of 59,460 containing 528 amino acids a nd displays much similarity to elongation factor EF-G, a GTP binding p rotein necessary for ribosomal translocation, and other GTP binding pr oteins known or thought to interact with the ribosome.