RESUMO
The RimM protein in Escherichia coli is associated with free 30S ribosomal subunits but not with 70S ribosomes. A DeltarimM mutant is defective in 30S maturation and accumulates 17S rRNA. To study the interaction of RimM with the 30S and its involvement in 30S maturation, RimM amino acid substitution mutants were constructed. A mutant RimM (RimM-YY-->AA), containing alanine substitutions for two adjacent tyrosines within the PRC beta-barrel domain, showed a reduced binding to 30S and an accumulation of 17S rRNA compared to wild-type RimM. The (RimM-YY-->AA) and DeltarimM mutants had significantly lower amounts of polysomes and also reduced levels of 30S relative to 50S compared to a wild-type strain. A mutation in rpsS, which encodes r-protein S19, suppressed the polysome- and 16S rRNA processing deficiencies of the RimM-YY-->AA but not that of the DeltarimM mutant. A mutation in rpsM, which encodes r-protein S13, suppressed the polysome deficiency of both rimM mutants. Suppressor mutations, found in either helices 31 or 33b of 16S rRNA, improved growth of both the RimM-YY-->AA and DeltarimM mutants. However, they suppressed the 16S rRNA processing deficiency of the RimM-YY-->AA mutant more efficiently than that of the DeltarimM mutant. Helices 31 and 33b are known to interact with S13 and S19, respectively, and S13 is known to interact with S19. A GST-RimM but not a GST-RimM(YY-->AA) protein bound strongly to S19 in 30S. Thus, RimM likely facilitates maturation of the region of the head of 30S that contains S13 and S19 as well as helices 31 and 33b.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Proteínas Ribossômicas/química , Proteínas Ribossômicas/metabolismo , Ribossomos/metabolismo , Alanina/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Proteínas de Bactérias/genética , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Glutationa Transferase/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação , Estrutura Terciária de Proteína , Processamento Pós-Transcricional do RNA , RNA Ribossômico 16S/genética , RNA Ribossômico 16S/metabolismo , Proteínas de Ligação a RNA , Proteínas Recombinantes/metabolismo , Proteínas Ribossômicas/genética , Homologia de Sequência de Aminoácidos , Tirosina/metabolismoRESUMO
The trmD operon is located at 56.7 min on the genetic map of the Escherichia coli chromosome and contains the genes for ribosomal protein (r-protein) S16, a 21-kDa protein (RimM, formerly called 21K), the tRNA (m1G37)methyltransferase (TrmD), and r-protein L19, in that order. Previously, we have shown that strains from which the rimM gene has been deleted have a sevenfold-reduced growth rate and a reduced translational efficiency. The slow growth and translational deficiency were found to be partly suppressed by mutations in rpsM, which encodes r-protein S13. Further, the RimM protein was shown to have affinity for free ribosomal 30S subunits but not for 30S subunits in the 70S ribosomes. Here we have isolated several new suppressor mutations, most of which seem to be located close to or within the nusA operon at 68.9 min on the chromosome. For at least one of these mutations, increased expression of the ribosome binding factor RbfA is responsible for the suppression of the slow growth and translational deficiency of a deltarimM mutant. Further, the RimM and RbfA proteins were found to be essential for efficient processing of 16S rRNA.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Escherichia coli , Escherichia coli/genética , Fatores de Alongamento de Peptídeos , Processamento Pós-Transcricional do RNA/genética , RNA Ribossômico 16S/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas Ribossômicas , Proteínas de Bactérias/fisiologia , Temperatura Baixa , Escherichia coli/crescimento & desenvolvimento , Dosagem de Genes , Genes Bacterianos/genética , Genes Supressores/genética , Óperon/genética , Elongação Traducional da Cadeia Peptídica/genética , RNA Bacteriano/análise , RNA Mensageiro/análise , Proteínas de Ligação a RNA/biossíntese , Fatores de Transcrição/genética , Fatores de Elongação da Transcrição , tRNA Metiltransferases/genéticaRESUMO
Previously, we showed that strains which have been deleted for the 21K gene (hereafter called yfjA), of the trmD operon, encoding a 21-kDa protein (21K protein) have an approximately fivefold-reduced growth rate in rich medium. Here we show that such mutants show an up to sevenfold reduced growth rate in minimal medium, a twofold-lower cell yield-to-carbon source concentration ratio, and a reduced polypeptide chain growth rate of beta-galactosidase. Suppressor mutations that increased the growth rate and translational efficiency of a delta yfjA mutant were localized to the 3' part of rpsM, encoding ribosomal protein S13. The 21K protein was shown to have affinity for free 30S ribosomal subunits but not for 70S ribosomes. Further, the 21K protein seems to contain a KH domain and a KOW motif, both suggested to be involved in binding of RNA. These findings suggest that the 21K protein is essential for a proper function of the ribosome and is involved in the maturation of the ribosomal 30S subunits or in translation initiation.
