Studies on the translation of genetic message : requirement for ribosomal protein S1 in natural mRNA translation

Abstract

The 30S ribosomal protein S1 of E. coli was previously shown to be identical with subunit I of QB replicase as well as with the protein synthesis interference factor "i". In vitro, S1 was shown to inhibit the translation of R17 RNA, and synthetic polynucleotides which contained a high proportion of pyrimidines. Initiation complex formation with R17 RNA as template was also reduced in the presence of S1. The purpose of the present work was to demonstrate a requirement for S1 in natural mRNA translation in a S1-free System. S1 is present on 30S ribosomal subunits and in the 1 M NH4C1 ribosomal wash. It was also detected in the high speed supernatant (S150 fraction). The protein was removed from 30S subunits by washing them extensively in the presence of 1 M NH4C1. Poly (C)-cellulose chromatography was utilized to remove S1 from the S150 fraction and to isolate 80-90% pure S1 from the 45-55% ammonium sulfate fraction of the 1 M NH4C1 ribosomal wash. The effect of SI on the translation of poly(U), poly(A) and R17 RNA was investigated in the S1-free system. S1 stimulated the translation of all messages. Optimal rates of amino acid incorporation were obtained when S1 was present at stoichiometric amounts in the assays. In the presence of excess S1, inhibition was observed with poly(U) or R17 RNA as template. Initiation complex formation with R17 RNA template exhibited a similar response, but S1 had no effect when AUG was used as template. Therefore, these studies demonstrate that S1 is required for normal ribosomal functions.