An investigation of the mechanisms controlling expression of the methionine regulon of E. coli K12 is proposed. Specifically, the properties of the metJ gene product, an aporepressor protein which in conjunction with S-adenosylmethionine, or derivatives thereof, becomes an active repressor which controls expression of all elements of the met regulon, will be analyzed. To accomplish this objective, deletion amber and temperature sensitive mutants of the metJ gene have been isolated. The in vivo effects of these alterations in metJ gene structure and function upon the expression of elements of the met regulon will be determined by two methods: (i) measurement of the specific activity of met gene products in crude cell extracts and (ii) determination of the in vivo levels of met regulon gene expression by RNA/DNA filter hybridization assays which employ characterized met restriction fragments as hybridization probes. The DNA sequence encoding the metJ gene product has been isolated and identified and will be used to analyze and characterize the gene product as it is synthesized from plasmid borne gene copies in UV-irridiated E. coli cells carrying recA mutations. DNA sequence determinations will be performed upon promoters of three met regulon transcription units which have been identified and characterized by this laboratory: (i) the metF unit; (ii) the metB(L?) unit and (iii) the metJ unit. The sequences of the three promoters will then be analyzed to identify any common structural and regulatory elements. The function(s) of components of these sequences will be evaluated by analysis of the in vitro interaction of purified E. coli RNA polymerase holoenzyme with isolated met regulon promoter sequences. Ultimately, the effects of the properly activated metJ gene product upon the interaction of RNA polymerase holoenzyme with the promoter structure will be evaluated.
| Collier, C D; Johnson, J R (1990) The Escherichia coli K-12 metJ193 allele contains a point mutation which alters the hydrophobic pocket responsible for in vitro binding of S-adenosylmethionine: effects on cell growth and induction of met regulon expression. J Bacteriol 172:3918-24 |
| Bala, G A; Collier, C D; Emmett, M R et al. (1989) Characterization of two mutant metJ proteins with reduced, temperature-dependent capacity to regulate Escherichia coli K-12 met regulon elements. J Bacteriol 171:4095-9 |
| Emmett, M R; Johnson, J R (1986) Control of metF gene expression in maxicell preparations of Escherichia coli K-12: reversible action of the metJ protein and effect of vitamin B12. J Bacteriol 168:1491-4 |
