In vitro synthesis of the tryptophan operon leader peptides of Escherichia coli, Serratia marcescens, and Salmonella typhimurium.

Abstract

We used an in vitro DNA-dependent proteinsynthesizing system to demonstrate de novo synthesis of the leader peptide specified by the tryptophan (trp) operons of several bacterial species. Peptide synthesis was directed by self-ligated short restriction fragments containing the trp promoter and leader regions. Synthesis of leader peptides was established by demonstrating that they were labeled in vitro only by those amino acids predicted to be present in the peptides. Leader peptide synthesis was abolished by the addition of the Escherichia coli trp repressor. The E. coli trp leader peptide was found to be extremely labile in vitro; it had a half-life of 3-4 min. In a highly purified DNAdependent peptide-synthesizing system, synthesis of the di-and tripeptides predicted from the Salmonella typhimurium trp operon leader sequence, fMet-Ala and fMet-Ala-Ala, also was observed. Using this dipeptide synthesis system, we demonstrated that translation initiation at the ribosome binding site used for trp leader peptide synthesis was reduced 10-fold when the transcript contained a segment complementary to the ribosome binding site. Many of the amino acid biosynthetic operons of bacteria are regulated by attenuation. Each of these operons has a transcribed leader region containing a short peptide-encoding segment rich in codons for the regulatory amino acid(s) (1-10). The presumptive leader peptides specified by these coding regions have been sought but have eluded detection. Nevertheless, gene fusion studies have demonstrated that the ribosome binding site of the trp operon leader region is an efficient site for translation initiation (11,12). We considered two explanations for our inability to detect the trp leader peptide. First, because synthesis of the peptide but not its survival was presumably of regulatory significance, we thought that the peptide might be rapidly degraded both in vivo and in vitro (11, 13). Second, scrutiny of the sequences of the trp leader transcripts of seven enteric bacterial species revealed that the distal segment of each leader transcript was complementary to the leader peptide ribosome binding site and conceivably could pair with that site and prevent multiple rounds of synthesis of the trp leader peptide (refs. 14-16; unpublished observations). The approach that was successful in the initial detection of the trp leader peptide, described herein, was suggested by Roberto Kolter of our laboratory.We performed in vitro coupled transcription/translation analyses, using as template a short restriction fragment containing the trp promoter-operator and leader peptide-encoding region and lacking the distal portion of the trp leader region that is complementary to the leader peptide ribosome binding marscens, and S. typhimurium. Nucleotide numbering is from the site oftranscription initiation (residue + 1). Negative numbers indicate the number ofbase pairs preceding the transcription start point. Numbers withinparentheses indicate that the exact site is notknown. TheE. coli template has a...

Translation of a synthetic two-cistron mRNA in Escherichia coli.

“…To reconcile these observations, it has been proposed that translation is inhibited when local secondary structures form with regions containing the SD sequence and/or the AUG start codon such that the ribosomes cannot initiate translation (20)(21)(22)(23)(24)(25). The formation of such secondary structures may explain failures to express methionyl bovine growth hormone (Met-bGH) with its native codons at high levels (26,27).…”

Overexpression of the dctA gene in Rhizobium meliloti: effect on transport of C4 dicarboxylates and symbiotic nitrogen fixation

“…We chose the trp promoter since it is well established as a strong promoter in E. coli and S. typhirnurium (Das et al 1983;Nichols and Yanofsky 1983) and has been used to cause gene overexpression in R. meliloti (Egelhoff and Long 1985). In E. coli, the trp operon is regulated by a repressor through an operator associated with the promoter (trpPO), but this regulation appears to be absent in R. meliloti.…”

Leader Peptide-mediated Transcriptional Attenuation of Lysine Biosynthetic Gene Cluster in Thermus thermophilus

“…The presence of a similar putative 5Ј-leader peptide has been suggested in regulation of amino acid biosynthesis, however, it has not been identified experimentally, except in a few cases (39). Furthermore, the mRNA for the lysine biosynthetic genes starts from a position only 3 bp upstream of the putative translational initiation codon of hcs leader peptide, suggesting a Shine-Dalgarno sequence-independent ribosome recognition of the translational initiation site, which can be seen only in some cases (40).…”