tRNA(Trp) translation of leader peptide codon 12 and other factors that regulate expression of the tryptophanase operon.

Tryptophanase (tna) operon expression in Escherichia coli is induced by tryptophan. This response is mediated by features of a 319-base-pair leader region preceding the major structural genes of the operon. Translation of the coding region (tnaC) for a 24-amino-acid leader peptide is essential for induction. We have used site-directed mutagenesis to investigate the role of the single Trp codon, at position 12 in tnaC, in regulation of the operon. Codon 12 was changed to either a UAG or UGA stop codon or to a CGG arginine codon. Induction by tryptophan was eliminated by any of these changes. Studies with suppressor tRNAs indicated that tRNA(Trp) translation of codon 12 in tnaC is essential for induction of the operon. Reduction of tna expression by a miaA mutation supports a role for translation by tRNA(Trp) in regulation of the operon. Frameshift mutations and suppression that allows translation of tnaC to proceed beyond the normal stop codon result in constitutive tna operon expression. Deletion of a potential site for Rho factor utilization just beyond tnaC also results in partial constitutive expression. These studies suggest possible models for tryptophan induction of tna operon expression involving tRNA(Trp)-mediated frame shifting or readthrough at the tnaC stop codon.     

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.     

Cooperation between the chloroplast psbA 5′‐untranslated region and coding region is important for translational initiation: the chloroplast translation machinery cannot read a human viral gene coding region

Chloroplast mRNA translation is regulated by the 5′‐untranslated region (5′‐UTR). Chloroplast 5′‐UTRs also support translation of the coding regions of heterologous genes. Using an in vitro translation system from tobacco chloroplasts, we detected no translation from a human immunodeficiency virus tat coding region fused directly to the tobacco chloroplast psbA 5′‐UTR. This lack of apparent translation could have been due to rapid degradation of mRNA templates or synthesized protein products. Replacing the psbA 5′‐UTR with the E. coli phage T7 gene 10 5′‐UTR, a highly active 5′‐UTR, and substituting synonymous codons led to some translation of the tat coding region. The Tat protein thus synthesized was stable during translation reactions. No significant degradation of the added tat mRNAs was observed after translation reactions. These results excluded the above two possibilities and confirmed that the tat coding region prevented its own translation. The tat coding region was then fused to the psbA 5′‐UTR with a cognate 5′‐coding segment. Significant translation was detected from the tat coding region when fused after 10 or more codons. That is, translation could be initiated from the tat coding region once translation had started, indicating that the tat coding region inhibits translational initiation but not elongation. Hence, cooperation/compatibility between the 5′‐UTR and its coding region is important for translational initiation.