Factors modulating transcription and translation in vitro of ribosomal protein S20 and isoleucyl-tRNA synthetase from Escherichia coli.

Abstract

The DNA-dependent protein-synthesizing system developed by Zubay [Zubay, G. (1973) Annu. Rev. Genet. 7, 267--287] was optimized for the transcription and translation of genes from the 0.5-min region of the Escherichia coli chromosome carried by transducing lambda phages. The E. coli gene products synthesized were isoleucyl tRNA synthetase, ribosomal protein S20, dihydrodipicolinic acid reductase and (possibly) the two subunits carbamoyl-phosphate synthetase. Formation of ribosomal protein S20 is specifically stimulated by the addition of 16-S rRNA and not by 5-S or 23-S rRNA. 16-S rRNA increases the rate of S20 synthesis, the final yield of product depends on the duration of persistence of the RNA added. Addition of 16-S rRNA to the separate transcription and translation systems showed that it is the translation of the S20 mRNA which is enhanced. Furthermore, S20 synthesis is stimulated more than fourfold when concomitant synthesis of rRNA occurs from a plasmid carrying an rrn transcriptional unit. The results described are explained in terms of a model which suggests that ribosomal protein S20 feedback inhibits its synthesis at the translational level and that removal of S20 into ribosomal assembly (i.e. binding to 16-S rRNA) releases inhibition. The model postulates a direct link between synthesis of ribosomal RNA and ribosomal protein and between the rates of ribosomal assembly and ribosomal protein synthesis. The stimulatory effect of guanosine 3'-diphosphate 5'-diphosphate on isoleucyl-tRNA synthetase formation and its inhibition of the synthesis of ribosomal protein S20 in vitro occurs at the level of transcription. Its relevance in vivo, however, remains to be demonstrated. Formation of isoleucyl-tRNA synthetase in vitro is not influenced either by the addition of a surplus of purified enzyme nor by the limitation of protein synthesis by the addition of anti-(isoleucyl-tRNA synthetase) serum. There is no evidence, therefore, that isoleucyl-tRNA synthetase is autogenously regulated.