Mutations in the leader sequence and initiation codon of the gene for ribosomal protein S20 (rpsT) affect both translational efficiency and autoregulation.

We have transferred the complete structural gene and part of the leader for ribosomal protein S20 of Escherichia coli to a controllable expression vector and have used oligonucleotide-directed mutagenesis to create mutations in the untranslated leader of the plasmid-borne gene. We have assayed for posttranscriptional regulation of the synthesis of S20 after inducing transcription of the mutant S20 mRNA from the expression vector. We found that two mutations lead to loss of feedback control of S20 synthesis: (i) a change of the initiation codon from UUG to AUG and (ii) a replacement of part of the S20 leader with a nonhomologous sequence including an AUG initiation codon. These mutations also lead to increases in both the intrinsic translational efficiency of the plasmid-encoded S20 mRNA in vitro and its half-life in vivo. A double mutation (GA to CT) at residues -3 and -4 relative to the initiation codon does not result in overproduction of S20. Rather, it reduces translational efficiency in vitro and mRNA stability in vivo. Our results demonstrate the fundamental importance of the UUG initiation codon in mediating autogenous repression of S20 synthesis.

Tandem promoters in the gene for ribosomal protein S20.

Examination of the nucleotide sequence of the gene for ribosomal protein S20 (rpsT) of Escherichia coli suggested the presence of two promoters ("sites 1 and 2") separated by 90 base pairs (Mackie, G. A. (1981) J. Biol. Chem. 256, 8177-8182). We have investigated the properties of purified or cloned DNA fragments containing one or other or both these sites for their ability to promote transcription in vivo and in vitro. In reactions in vitro containing DNA and purified RNA polymerase as the sole macromolecular components, both sites 1 and 2 act as promoters directing the synthesis of "runoff" transcripts. The 5' termini of such transcripts have been determined by direct sequencing or by identification of the 5' terminal nucleoside 5'-triphosphate, 3'-monophosphate. In site 1, the major transcript initiates with GTP at residue 141 in the DNA sequence. A minor start occurs at residue 142 and uses CTP as the initiating nucleotide. In site 2, the major transcript (approximately 55% of all initiations in site 2) initiates with CTP at residue 232 while minor transcripts, each comprising approximately 20% of the total, initiate at residues 231 and 233 with GTP and CTP, respectively. In four methods of assay which reflect to varying extents the usage of promoters in vivo, site 1 is responsible for 10-30% of the total transcription of the gene for S20 and site 2 the remainder. Sites 1 and 2 appear to act independently and additively in assays based on the rate of synthesis of S20 in a system for coupled transcription and translation. Together, the two promoters for S20 are from 10-25-fold more active than the fully induced lac operon promoter.

Expression of the gene for ribosomal protein S20: effects of gene dosage.

We have exploited the properties of three different plasmids which carry the gene for Escherichia coli ribosomal protein S20 (rpsT) to test the effects of gene dosage on the expression of rpsT. Over a range of total copies of rpsT of 1 to 58 per haploid genome equivalent, the rate of incorporation of uridine during a 30-s pulse into RNA annealing to either of two specific probes for S20 mRNA increased essentially in proportion to copy number. In contrast, the rate of synthesis of S20 protein increased no more than 2.1-fold at the highest copy number. We conclude, in contrast to an earlier report (D. Geyl, and A. Böck, Mol. Gen. Genet. 154:327-334, 1977), that the synthesis of S20 is regulated at a posttranscriptional step. We propose that S20 itself is the regulatory agent and that binding of S20 to its own mRNA in regions homologous in structure with 16S rRNA can account for our results.

Identification of gene products from cloned fragments of the left arm of lambda dapB2.

A set of recombinant plasmids encompassing the bacterial substitution in lambda dapB2 (Mackie, G. A. (1980) J. Biol. Chem. 255, 8928--8935) has been characterized genetically by complementation and biochemically by an analysis of the proteins encoded by individual plasmids in vitro. In addition to a cluster of four genes which includes the dapB locus, a total of seven gene products has been identified. One of these is ribosomal protein S20, whose gene (rpsT) has been localized to a 0.56-kilobase segment of DNA bounded by HindIII and HindII sites. Positive identification of this gene on plasmids pGM9 and pGP2 has been achieved by analysis of the products encoded by these plasmids in vitro and by the ability of pGM9 to complement a strain lacking S20 among its 30S subunit proteins. A second gene is that for isoleucyl tRNA synthetase (ileS). Its presence on pGM21 has been ascertained by the latter's ability to direct the synthesis of a protein in vitro with the size anticipated for this gene product. Extracts of cells harbouring this plasmid also exhibit greater isoleucyl tRNA synthetase activity than parental extracts. A third gene is at least 1.3 kilobases distant from rpsT and encodes a protein of 24 000 molecular weight, of unknown function. This locus is in turn about 6.5 kilobases from lambda gene E. The latter gene, and several others, all of which likely derive from lambda DNA are carried on a 5.2-kilobase fragment inserted in the plasmid pGM11 which includes the junction between phage and bacterial sequences on the left side of lambda dapB2. This fragment of DNA is inserted into the vector in a manner which permits the coupled transcription and translation of lambda genes D and E in vitro, despite the absence of their natural promoter. The most striking feature of the organization of the bacterial genes on lambda dapB2 is the clustering of genes for polypeptides in the right half of the bacterial substitution in contrast to the apparent minimal use of the coding potential in the left half of the substitution.