Translation of the genetic message, iii. Formylmethionine as initiator of proteins programed by polycistronic messenger RNA.

Polyacrylamide gel electrophoresis of the virus-specific proteins synthesized in vivo upon infection of E. coli with phage AMS21 2 yields three main fractions designated as proteins I, II, and III, of which protein III is coat protein. Experimerits with "amber" mutants indicate that protein JI is a viral RNA synlthetase, whereas protein I12 ' is a "maturation" protein.4 Translation of the RNA of coliphages by cell-free systems of E. coli gives rise to coat and other virus-specific proteins.5-8 Two of these proteins have been characterized as RNA synthetase and coat protein.8 The involvement of formylmethionine in initiation of coat protein synthesis9' 10 raises the question whether other virus-specific proteins are similarly initiated. There are some indications that this is the case.9' 11 The present work was undertaken in order (a) to further characterize the proteins formed upon translation of the MiS2 genome in vitro, and (b) to determine whether formylmethionine is the chain initiator of proteins other than coat. For characterization of the proteins synthesized in vitro, the C'4-labeled proteins formed by a cell-free E. coli system with i\1S2 RNA as messenger were coelectrophoresed with H3-labeled proteins produced by 1VIS2-infected, actinomycin-treated E. coli spheroplasts. The in vivo and in vitro proteins I and III were qualitatively identical but the in vitro system produced protein II to a much lesser extent, if at all. Moreover, the presence of one or more peaks, with a mobility intermediate between that of proteins II and III, suggested that unfinished or degraded peptides had been formed. In order to determine whether formylmethionine is involved in the initiation of both coat protein and protein I, in vitro incubations were conducted with H'-formyltetrahydrofolic acid and C'4-methionine, and the proteins isolated by polyacrylamide gel electrophoresis. The two proteins contained HI and C'4 label and both yielded formylmethionine upon digestion with pronase. Materials and Methods.-Ribosomes, supernatant, and initiation factors: Ribosomes from E. coli Q13 (a mutant lacking ribonuclease I and polynucleotide phosphorylase) were purified by chromatography on O-(diethylaminoethyl) cellulose (DEAE-cellulose) as in previous work.'2 Supernatant fractions were prepared from E. coli Q13 as described earlier,"3 except that only the upper two thirds of the supernatant was used. Chain initiation factors (F1 and F2), required for translation of natural messenger when purified ribosomes are used, were prepared from E. coli Q13 as previously outlined.'4 They were free of nuclease activity. Other preparations: RNA from phage MS2 was purified by the method of Strauss and Sinsheimer."5 Sucrose density gradient centrifugation of the RNA showed a single peak at 28S. The preparation was kept frozen for about 3 weeks before use. H'-FTHF'6 was prepared'7 with H3formate using formyltetrahydrofolic acid (FTHF) synthetase kindly provided by Dr. J. C. Rabinowitz, University of California, Berkeley. The H'-FTHF was purified on a column of Whatman cellulose powder."8 Formylmethionyl-alanine was prepared by formylation of methionyl-alanine.19 The reaction mixture was passed through a column of Dowex 50, H+ form, and the formylmethionyl-alanine was eluted with water.