Abstract
Expression of the tryptophanase (tna) operon of Escherichia coli is regulated by catabolite repression and by tryptophan-induced transcription antitermination at Rho-dependent termination sites in the leader region of the operon. Tryptophan induction is dependent on translation of a short leader peptide coding region, tnaC, that contains a single, crucial tryptophan codon. Recent studies suggest that during induction, the TnaC leader peptide acts in cis on the translating ribosome to inhibit its release at the tnaC stop codon. In the present study we use a tnaC-UGA-'lacZ construct lacking the tnaC-tnaA spacer region to analyze the effect of TnaC synthesis on the behavior of the ribosome that translates tnaC. The tnaC-UGA-'lacZ construct is not expressed significantly in the presence or absence of inducer. However, it is expressed in the presence of UGA suppressors, or when the structural gene for polypeptide release factor 3 is disrupted, or when wild-type tRNATrP is overproduced. In each situation, tnaC-UGA-'lacZ expression is reduced appreciably by the presence of inducing levels of tryptophan. Replacing the tnaC UGA stop codon with a sense codon allows considerable expression, which is also reduced, although to a lesser extent, by the addition of tryptophan. Inhibition by tryptophan is not observed when Trp codon 12 of tnaC is changed to a Leu codon. Overexpression of tnaC in trans from a multicopy plasmid prevents inhibition of expression by tryptophan. These results support the hypothesis that the TnaC leader peptide acts in cis to alter the behavior of the translating ribosome.
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- Alexieva Z., Duvall E. J., Ambulos N. P., Jr, Kim U. J., Lovett P. S. Chloramphenicol induction of cat-86 requires ribosome stalling at a specific site in the leader. Proc Natl Acad Sci U S A. 1988 May;85(9):3057–3061. doi: 10.1073/pnas.85.9.3057. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bolivar F., Rodriguez R. L., Greene P. J., Betlach M. C., Heyneker H. L., Boyer H. W., Crosa J. H., Falkow S. Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene. 1977;2(2):95–113. [PubMed] [Google Scholar]
- Botsford J. L., DeMoss R. D. Catabolite repression of tryptophanase in Escherichia coli. J Bacteriol. 1971 Jan;105(1):303–312. doi: 10.1128/jb.105.1.303-312.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Casadaban M. J., Chou J., Cohen S. N. In vitro gene fusions that join an enzymatically active beta-galactosidase segment to amino-terminal fragments of exogenous proteins: Escherichia coli plasmid vectors for the detection and cloning of translational initiation signals. J Bacteriol. 1980 Aug;143(2):971–980. doi: 10.1128/jb.143.2.971-980.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Deeley M. C., Yanofsky C. Nucleotide sequence of the structural gene for tryptophanase of Escherichia coli K-12. J Bacteriol. 1981 Sep;147(3):787–796. doi: 10.1128/jb.147.3.787-796.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Dorman C. J., Foster T. J. Posttranscriptional regulation of the inducible nonenzymatic chloramphenicol resistance determinant of IncP plasmid R26. J Bacteriol. 1985 Jan;161(1):147–152. doi: 10.1128/jb.161.1.147-152.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Edwards R. M., Yudkin M. D. Location of the gene for the low-affinity tryptophan-specific permease of Escherichia coli. Biochem J. 1982 May 15;204(2):617–619. doi: 10.1042/bj2040617. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gish K., Yanofsky C. Evidence suggesting cis action by the TnaC leader peptide in regulating transcription attenuation in the tryptophanase operon of Escherichia coli. J Bacteriol. 1995 Dec;177(24):7245–7254. doi: 10.1128/jb.177.24.7245-7254.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gish K., Yanofsky C. Inhibition of expression of the tryptophanase operon in Escherichia coli by extrachromosomal copies of the tna leader region. J Bacteriol. 1993 Jun;175(11):3380–3387. doi: 10.1128/jb.175.11.3380-3387.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gollnick P., Yanofsky C. tRNA(Trp) translation of leader peptide codon 12 and other factors that regulate expression of the tryptophanase operon. J Bacteriol. 1990 Jun;172(6):3100–3107. doi: 10.1128/jb.172.6.3100-3107.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gu Z., Harrod R., Rogers E. J., Lovett P. S. Anti-peptidyl transferase leader peptides of attenuation-regulated chloramphenicol-resistance genes. Proc Natl Acad Sci U S A. 1994 Jun 7;91(12):5612–5616. doi: 10.1073/pnas.91.12.5612. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gu Z., Rogers E. J., Lovett P. S. Peptidyl transferase inhibition by the nascent leader peptide of an inducible cat gene. J Bacteriol. 1993 Sep;175(17):5309–5313. doi: 10.1128/jb.175.17.5309-5313.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hirsh D. Tryptophan transfer RNA as the UGA suppressor. J Mol Biol. 1971 Jun 14;58(2):439–458. doi: 10.1016/0022-2836(71)90362-7. [DOI] [PubMed] [Google Scholar]
- Hopkins F. G., Cole S. W. A contribution to the chemistry of proteids: Part II. The constitution of tryptophane, and the action of bacteria upon it. J Physiol. 1903 Jun 15;29(4-5):451–466. doi: 10.1113/jphysiol.1903.sp000968. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kamath A. V., Yanofsky C. Characterization of the tryptophanase operon of Proteus vulgaris. Cloning, nucleotide sequence, amino acid homology, and in vitro synthesis of the leader peptide and regulatory analysis. J Biol Chem. 1992 Oct 5;267(28):19978–19985. [PubMed] [Google Scholar]
- Kamath A. V., Yanofsky C. Roles of the tnaC-tnaA spacer region and Rho factor in regulating expression of the tryptophanase operon of Proteus vulgaris. J Bacteriol. 1997 Mar;179(5):1780–1786. doi: 10.1128/jb.179.5.1780-1786.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kawasaki K., Yokota A., Oita S., Kobayashi C., Yoshikawa S., Kawamoto S., Takao S., Tomita F. Cloning and characterization of a tryptophanase gene from Enterobacter aerogenes SM-18. J Gen Microbiol. 1993 Dec;139(12):3275–3281. doi: 10.1099/00221287-139-12-3275. [DOI] [PubMed] [Google Scholar]
- Kazarinoff M. N., Snell E. E. Essential arginine residues in tryptophanase from Escherichia coli. J Biol Chem. 1977 Nov 10;252(21):7598–7602. [PubMed] [Google Scholar]
- Lovett P. S., Rogers E. J. Ribosome regulation by the nascent peptide. Microbiol Rev. 1996 Jun;60(2):366–385. doi: 10.1128/mr.60.2.366-385.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lovett P. S. Translational attenuation as the regulator of inducible cat genes. J Bacteriol. 1990 Jan;172(1):1–6. doi: 10.1128/jb.172.1.1-6.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Mikuni O., Ito K., Moffat J., Matsumura K., McCaughan K., Nobukuni T., Tate W., Nakamura Y. Identification of the prfC gene, which encodes peptide-chain-release factor 3 of Escherichia coli. Proc Natl Acad Sci U S A. 1994 Jun 21;91(13):5798–5802. doi: 10.1073/pnas.91.13.5798. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Monro R. E., Marcker K. A. Ribosome-catalysed reaction of puromycin with a formylmethionine-containing oligonucleotide. J Mol Biol. 1967 Apr 28;25(2):347–350. doi: 10.1016/0022-2836(67)90146-5. [DOI] [PubMed] [Google Scholar]
- NEWTON W. A., SNELL E. E. CATALYTIC PROPERTIES OF TRYPTOPHANASE, A MULTIFUNCTIONAL PYRIDOXAL PHOSPHATE ENZYME. Proc Natl Acad Sci U S A. 1964 Mar;51:382–389. doi: 10.1073/pnas.51.3.382. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Raftery L. A., Egan J. B., Cline S. W., Yarus M. Defined set of cloned termination suppressors: in vivo activity of isogenetic UAG, UAA, and UGA suppressor tRNAs. J Bacteriol. 1984 Jun;158(3):849–859. doi: 10.1128/jb.158.3.849-859.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Richardson L. V., Richardson J. P. Rho-dependent termination of transcription is governed primarily by the upstream Rho utilization (rut) sequences of a terminator. J Biol Chem. 1996 Aug 30;271(35):21597–21603. doi: 10.1074/jbc.271.35.21597. [DOI] [PubMed] [Google Scholar]
- Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sarkar G., Sommer S. S. The "megaprimer" method of site-directed mutagenesis. Biotechniques. 1990 Apr;8(4):404–407. [PubMed] [Google Scholar]
- Simons R. W., Houman F., Kleckner N. Improved single and multicopy lac-based cloning vectors for protein and operon fusions. Gene. 1987;53(1):85–96. doi: 10.1016/0378-1119(87)90095-3. [DOI] [PubMed] [Google Scholar]
- Stewart V., Landick R., Yanofsky C. Rho-dependent transcription termination in the tryptophanase operon leader region of Escherichia coli K-12. J Bacteriol. 1986 Apr;166(1):217–223. doi: 10.1128/jb.166.1.217-223.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Stewart V., Yanofsky C. Evidence for transcription antitermination control of tryptophanase operon expression in Escherichia coli K-12. J Bacteriol. 1985 Nov;164(2):731–740. doi: 10.1128/jb.164.2.731-740.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Stewart V., Yanofsky C. Role of leader peptide synthesis in tryptophanase operon expression in Escherichia coli K-12. J Bacteriol. 1986 Jul;167(1):383–386. doi: 10.1128/jb.167.1.383-386.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
- VOGEL H. J., BONNER D. M. Acetylornithinase of Escherichia coli: partial purification and some properties. J Biol Chem. 1956 Jan;218(1):97–106. [PubMed] [Google Scholar]
- Vogel U., Jensen K. F. Effects of the antiterminator BoxA on transcription elongation kinetics and ppGpp inhibition of transcription elongation in Escherichia coli. J Biol Chem. 1995 Aug 4;270(31):18335–18340. doi: 10.1074/jbc.270.31.18335. [DOI] [PubMed] [Google Scholar]
- Watanabe T., Snell E. E. Reversibility of the tryptophanase reaction: synthesis of tryptophan from indole, pyruvate, and ammonia. Proc Natl Acad Sci U S A. 1972 May;69(5):1086–1090. doi: 10.1073/pnas.69.5.1086. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Yanofsky C., Horn V. Bicyclomycin sensitivity and resistance affect Rho factor-mediated transcription termination in the tna operon of Escherichia coli. J Bacteriol. 1995 Aug;177(15):4451–4456. doi: 10.1128/jb.177.15.4451-4456.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Yanofsky C., Horn V., Nakamura Y. Loss of overproduction of polypeptide release factor 3 influences expression of the tryptophanase operon of Escherichia coli. J Bacteriol. 1996 Jul;178(13):3755–3762. doi: 10.1128/jb.178.13.3755-3762.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Yarus M., McMillan C., 3rd, Cline S., Bradley D., Snyder M. Construction of a composite tRNA gene by anticodon loop transplant. Proc Natl Acad Sci U S A. 1980 Sep;77(9):5092–5096. doi: 10.1073/pnas.77.9.5092. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Zwiefka A., Kohn H., Widger W. R. Transcription termination factor rho: the site of bicyclomycin inhibition in Escherichia coli. Biochemistry. 1993 Apr 13;32(14):3564–3570. doi: 10.1021/bi00065a007. [DOI] [PubMed] [Google Scholar]