Roles of the tnaC-tnaA spacer region and Rho factor in regulating expression of the tryptophanase operon of Proteus vulgaris.
To localize the DNA regions responsible for basal-level and induced expression of the tryptophanase (tna) operon of Proteus vulgaris, short deletions were introduced in the 115-bp spacer region separating tnaC, the leader peptide coding region, from tnaA. Deletions were incorporated into a tnaA'-'lacZ reporter construct containing the intact tna promoter-leader region. Expression was examined in Escherichia coli. Deletions that removed 28 to 30 bp from the region immediately following tnaC increased basal-level expression about threefold and allowed threefold induction by 1-methyltryptophan. A deletion removing 34 bp from the distal segment of the leader permitted basal and induced expression comparable to that of the parental construct. The mutant with the largest spacer deletion, 89 bp, exhibited a 30-fold increase in basal-level expression, and most importantly, inducer presence reduced operon expression by ca. 60%. Replacing the tnaC start codon or replacing or removing Trp codon 20 of tnaC of this deletion derivative eliminated inducer inhibition of expression. These findings suggest that the spacer region separating tnaC and tnaA is essential for Rho action. They also suggest that juxtaposition of the tnaC stop codon and the tnaA ribosome binding site in the 89-bp deletion derivative allows the ribosome that has completed translation of tnaC to inhibit translation initiation at the tnaA start codon when cells are exposed to inducer. These findings are consistent with results in the companion article that suggest that inducer allows the TnaC peptide to inhibit ribosome release at the tnaC stop codon.
Kamath
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AV
Department of Molecular Sciences, Central Research Division, Pfizer Inc., Groton, Connecticut 06340, USA.
Yanofsky
C
C
D001426:Bacterial Proteins / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000494:pharmacology; D003062:Codon; D018388:Codon, Terminator; D004275:DNA, Ribosomal / Q000235:genetics*; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D009876:Operon; D010957:Plasmids; D014176:Protein Biosynthesis; D011514:Proteus vulgaris / Q000201:enzymology / Q000235:genetics*; D012234:Rho Factor / Q000502:physiology*; D012270:Ribosomes / Q000378:metabolism; D017384:Sequence Deletion; D014364:Tryptophan / Q000235:genetics; D014368:Tryptophanase / Q000235:genetics*
D001426:Bacterial Proteins; D019086:Bridged Bicyclo Compounds, Heterocyclic; D003062:Codon; D018388:Codon, Terminator; D004275:DNA, Ribosomal; D029968:Escherichia coli Proteins; D012234:Rho Factor; C081473:tnaC protein, E coli; D014364:Tryptophan; D014368:Tryptophanase; C024504:bicozamycin