Analyses will continue on the molecular features of regulation of gene expression by transcription attenuation in the trp operon of Enterobacteria, the trp operon of Bacilli, and the tryptophanase (tna) operon of E. coli. Each of theses examples involves different components and events, but the consequences of the regulatory decision is the same - determination whether or not transcription will be terminated at a site or sites in the leader region of the respective operon. Studies with the trp operon of Enterobacteria will focus on the mechanism of basal level control, and will involve analysis of ribosome release at the leader peptide stop codon, an assessment of the contributions of the alternative transcript hairpin structures, and an examination of the importance of the position of the transcribing polymerase at the moment the translating ribosome dissociates from the leader stop codon. In addition, several aspects of transcription pausing will be investigated, including the role of the antiterminator hairpin as a pause signal. The importance of the design of the leader peptide coding region also will be investigated. Studies on attenuation in the trp operon of Bacilli will concentrate on characterizing the regulatory gene, mtr, and its polypeptide product. The mechanism of action of this RNA binding regulatory protein will be determined; specifically Mtr's interaction with its target sequence in the leader transcript will be examined. Studies will be performed to verify the initial observation that during growth in excess tryptophan translation of the trpE coding region of the read through transcript is inhibited. Studies with the tna operon will concentrate on the role of the leader peptide and its translation in regulation of transcription termination at Rho factor- dependent termination sites in the distal segment of the leader region. A newly discovered locus that confers constitutive expression on the operon in trans, will be further characterized. Analyses will be performed to determine if tryptophan activation of a presumed antitermination protein renders the transcribing polymerase refractory to termination. Studies with these attenuation systems should reveal fundamental features of transcription, and transcriptional and translational control. In addition, attenuation in the B. subtilis trp operon may serve as a prokaryotic model for attenuation in higher organisms.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37GM009738-30
Application #
3484083
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1979-05-01
Project End
1994-04-30
Budget Start
1992-05-01
Budget End
1993-04-30
Support Year
30
Fiscal Year
1992
Total Cost
Indirect Cost
Name
Stanford University
Department
Type
Schools of Arts and Sciences
DUNS #
800771545
City
Stanford
State
CA
Country
United States
Zip Code
94305
Babitzke, P; Stults, J T; Shire, S J et al. (1994) TRAP, the trp RNA-binding attenuation protein of Bacillus subtilis, is a multisubunit complex that appears to recognize G/UAG repeats in the trpEDCFBA and trpG transcripts. J Biol Chem 269:16597-604
Gish, K; Yanofsky, C (1993) Inhibition of expression of the tryptophanase operon in Escherichia coli by extrachromosomal copies of the tna leader region. J Bacteriol 175:3380-7
Babitzke, P; Yanofsky, C (1993) Reconstitution of Bacillus subtilis trp attenuation in vitro with TRAP, the trp RNA-binding attenuation protein. Proc Natl Acad Sci U S A 90:133-7
Kamath, A V; Yanofsky, C (1993) Sequence and characterization of mutT from Proteus vulgaris. Gene 134:99-102
Kamath, A V; Yanofsky, C (1992) 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 267:19978-85
Babitzke, P; Gollnick, P; Yanofsky, C (1992) The mtrAB operon of Bacillus subtilis encodes GTP cyclohydrolase I (MtrA), an enzyme involved in folic acid biosynthesis, and MtrB, a regulator of tryptophan biosynthesis. J Bacteriol 174:2059-64
Roesser, J R; Yanofsky, C (1991) The effects of leader peptide sequence and length on attenuation control of the trp operon of E.coli. Nucleic Acids Res 19:795-800
Landick, R; Yanofsky, C; Choo, K et al. (1990) Replacement of the Escherichia coli trp operon attenuation control codons alters operon expression. J Mol Biol 216:25-37
Roesser, J R; Yanofsky, C (1990) The RNA antiterminator causes transcription pausing in the leader region of the tryptophan operon. J Biol Chem 265:6055-60
Gollnick, P; Ishino, S; Kuroda, M I et al. (1990) The mtr locus is a two-gene operon required for transcription attenuation in the trp operon of Bacillus subtilis. Proc Natl Acad Sci U S A 87:8726-30

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