metF Gene Research Articles

Nucleotide sequence of metF, the E. coli structural gene for 5-10 methylene tetrahydrofolate reductase and of its control region.

The nucleotide sequence of the E.coli metF gene (888 nucleotides), coding for 5-10 methylene tetrahydrofolate reductase, has been determined. The metF gene product was identified in maxicells and found to be a protein of subunit molecular weight 33,000, in agreement with the size of the coding region. The starting point for metF transcription was determined by S1 nuclease mapping. No structural evidence was found for an attenuation mechanism regulating the independent metF transcriptional unit. Comparison of the regulatory region preceding the metF structural gene with the 5' flanking region of the metBL operon shows some homology spanning 24 nucleotides. These homologous sequences could be operator structures belonging to the two transcriptional units, metF and metBL, and recognized by the same regulatory protein.

Mutations affecting regulation of methionine biosynthetic genes isolated by use of met-lac fusions.

Fusions of the lac genes to the promoters of four structural genes in the methionine biosynthetic pathway, metA, metB, metE, and metF, were obtained by the use of the Mu d(Ap lac) bacteriophage. The levels of beta-galactosidase in these strains could be derepressed by growth under methionine-limiting conditions. Furthermore, growth in the presence of vitamin B12 repressed the synthesis of beta-galactosidase in strains containing a fusion of lacZ to the metE promoter, phi(metE'-lacZ+). Mutations affecting the regulation of met-lac fusions were generated by the insertion of Tn5. Tn5 insertions were obtained at the known regulatory loci metJ and metK. Interestingly, a significant amount of methionine adenosyltransferase activity remained in the metK mutant despite the fact that the mutation was generated by an insertion. Several Tn5-induced regulatory mutations were isolated by screening for high-level beta-galactosidase expression in a phi(metE'-lacZ+) strain in the presence of vitamin B12. Tn5 insertions mapping at the btuB (B12 uptake), metH (B12 dependent tetrahydropteroylglutamate methyltransferase), and metF (5,10-methylenetetrahydrofolate reductase) loci were obtained. The isolation of the metH mutant was consistent with previous suggestions that the metH gene product is required for the repression of metE by vitamin B12. The metF::Tn5 insertion was of particular interest since it suggested that a functional metf gene product was also needed for repression of metE by vitamin B12.