Factors Modulating Transcription and Translation in vitro of Ribosomal Protein S20 and Isoleucyl‐tRNA Synthetase from Escherichia coli

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Abstract
The DNA-dependent protein-synthesizing system developed by Zubay (1973) was optimized for the transcription and translation of genes from the 0.5 min region of the E. coli chromosome carried by transducing 2 phages. The E. coli gene products synthesized were isoleucly-tRNA synthetase (EC 6.1.15) ribosomal protein S20 dihydrodipicolinic acid reductase (EC 1.3.1.26) and (possibly) the 2 subunits of carbamoyl-phosphate synthetase [i.e., the heavy subunit (EC 6.3.4.16) and the small subunit (EC 6.3.5.5)]. Formation of ribosomal protein S20 was specifically stimulated by the addition of 16S rRNA and not by 5S or 23S rRNA 16S rRNA increased the rate of S20 synthesis, the final yield of product depends on the duration of persistence of the RNA added. Addition of 16S rRNA to the separate transcription and translation systems showed that it is the translation of the S20 mRNA which is enhanced. S20 synthesis is stimulated more than 4-fold when concomitant synthesis or rRNA occurs form a plasmid carrying an rrn transcriptional unit. A model is described 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 16S rRNA) releases inhibition. The model postulates a direct link between synthesis of rRNA 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 occurred at the level of transcription. Its relevance in vivo remains to be demonstrated. Formation of isoleucyl-tRNA synthetase in vitro was 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. Isoleucyl-tRNA synthetase is apparently not autogenously regulated.