RESUMO
The RimM protein in Escherichia coli is important for the in vivo maturation of 30S ribosomal subunits and a ΔrimM mutant grows poorly due to assembly and translational defects. These deficiencies are suppressed partially by mutations that increase the synthesis of another assembly protein, RbfA, encoded by the metY-nusA-infB operon. Among these suppressors are mutations in nusA that impair the NusA-mediated negative-feedback regulation at internal intrinsic transcriptional terminators of the metY-nusA-infB operon. We describe here the isolation of two new mutations, one in rpoB and one in rpoC (encoding the ß and ß' subunits of the RNA polymerase, respectively), that increase the synthesis of RbfA by preventing NusA from stimulating termination at the internal intrinsic transcriptional terminators of the metY-nusA-infB operon. The rpoB2063 mutation changed the isoleucine in position 905 of the ß flap-tip helix to a serine, while the rpoC2064 mutation duplicated positions 415 to 416 (valine-isoleucine) at the base of the ß' dock domain. These findings support previously published in vitro results, which have suggested that the ß flap-tip helix and ß' dock domain at either side of the RNA exit tunnel mediate the binding to NusA during transcriptional pausing and termination.
Assuntos
RNA Polimerases Dirigidas por DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Óperon/fisiologia , Fatores de Alongamento de Peptídeos/metabolismo , Fator de Iniciação 2 em Procariotos/metabolismo , Fatores de Transcrição/metabolismo , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica/fisiologia , Mutação , Óperon/genética , Fatores de Alongamento de Peptídeos/genética , Fator de Iniciação 2 em Procariotos/genética , Estrutura Terciária de Proteína , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Fatores de Transcrição/genética , Transcrição Gênica/fisiologia , Fatores de Elongação da TranscriçãoRESUMO
The in vivo assembly of ribosomal subunits requires assistance by auxiliary proteins that are not part of mature ribosomes. More such assembly proteins have been identified for the assembly of the 50S than for the 30S ribosomal subunit. Here, we show that the RimP protein (formerly YhbC or P15a) is important for the maturation of the 30S subunit. A rimP deletion (DeltarimP135) mutant in Escherichia coli showed a temperature-sensitive growth phenotype as demonstrated by a 1.2-, 1.5-, and 2.5-fold lower growth rate at 30, 37, and 44 degrees C, respectively, compared to a wild-type strain. The mutant had a reduced amount of 70S ribosomes engaged in translation and showed a corresponding increase in the amount of free ribosomal subunits. In addition, the mutant showed a lower ratio of free 30S to 50S subunits as well as an accumulation of immature 16S rRNA compared to a wild-type strain, indicating a deficiency in the maturation of the 30S subunit. All of these effects were more pronounced at higher temperatures. RimP was found to be associated with free 30S subunits but not with free 50S subunits or with 70S ribosomes. The slow growth of the rimP deletion mutant was not suppressed by increased expression of any other known 30S maturation factor.
Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Subunidades Ribossômicas Menores de Bactérias/metabolismo , Citoplasma/química , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Proteínas de Escherichia coli/genética , Deleção de Genes , Biossíntese de Proteínas , RNA Ribossômico 16S/metabolismo , Subunidades Ribossômicas Menores de Bactérias/química , TemperaturaRESUMO
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.