RESUMEN
The tRNA(m5U54)methyltransferase, whose structural gene is designated trmA, catalyzes the formation of 5-methyluridine in position 54 of all tRNA species in Escherichia coli. The synthesis of this enzyme has previously been shown to be both growth rate dependent and stringently regulated, suggesting regulatory features similar to those of rRNA. We have determined the complete nucleotide sequence of the trmA operon in E. coli and the sequence of the trmA promoter region in Salmonella typhimurium and also analyzed the transcriptional regulation of the gene. The trmA and the btuB (encoding the vitamin B12 outer membrane receptor protein) promoters are divergent promoters separated by 102 bp between the transcriptional start sites. The trmA promoters of both E. coli and S. typhimurium share promoter elements with the rRNA P1 promoter. The sequence downstream from the -10 region of the trmA promoter is homologous to the discriminatory region found in stringently regulated promoters. Next to and upstream from the -10 region is a sequence, TCCC, in the trmA promoter that is present in all of the seven rRNA P1 promoters and in some tRNA promoters but not in any other sigma 70 promoter. However, a similar motif is also found in promoters transcribed by the heat shock sigma factor sigma 32. The trmA gene is transcribed as a monocistronic operon, and the 3' end of the transcript is shown to be located downstream from a dyad symmetry region not followed by a poly(U) stretch. Using a trmA-cat operon fusion, we show that the growth rate-dependent regulation of trmA resembles that of rRNA and operates at the level of transcription.
Asunto(s)
Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos , Familia de Multigenes , Regiones Promotoras Genéticas , ARN Ribosómico/genética , ARN de Transferencia/metabolismo , ARNt Metiltransferasas/genética , Secuencia de Aminoácidos , Secuencia de Bases , Escherichia coli/enzimología , Escherichia coli/crecimiento & desarrollo , Datos de Secuencia Molecular , Plásmidos , Mapeo Restrictivo , Salmonella typhimurium/genética , Homología de Secuencia de Ácido Nucleico , Transcripción GenéticaRESUMEN
The enzyme catalyzing the formation of 5-methyluridine (ribothymidine) in tRNA of Escherichia coli is tRNA (uracil-5)-methyltransferase (EC 2.1.1.35). A 2.8-kilobase EcoRI chromosomal DNA fragment contains trmA, the structural gene for this enzyme. Subcloning, transcription in vitro, Tn5 insertion mutagenesis, and transcriptional fusion experiments were performed to establish the gene organization of the trmA region on the E. coli chromosome. trmA is a monocistronic operon. The trmA promoter was localized by in vitro experiments, and the direction of transcription was shown to be counterclockwise on the standard E. coli K-12 chromosomal map. The level of transcription of trmA in vitro and the expression of protein in minicells equal those of the bla gene of plasmid pBR322.