RESUMO
Escherichia coli RNase G, encoded by the rng gene, is involved in both the processing of 16S rRNA precursor and the degradation of adhE mRNA. Consequently, defects in RNase G result in elevation of AdhE levels. Furthermore, the adhR430 mutant strain, DC430, is reported to overproduce the AdhE protein in a manner dependent on the adhC81 mutation. We found that overproduction of AdhE by DC430 was reversed to wild-type levels by introduction of a plasmid carrying the wild-type allele of rng. Mapping by P1-phage-mediated transduction also indicated that a mutation involved in AdhE overproduction was located around the rng region in DC430. DNA sequencing of the rng region revealed that DC430 indeed had a mutation in the rng gene: a G1022 to A transition that caused substitution of Gly341 with Ser and which was named rng430. This lies in the highly conserved region of the RNase E/RNase G family, called high similarity region 2 (HSR2). However, very interestingly, rng430 mutant strains did not accumulate the 16.3S precursor of 16S rRNA unlike rng::cat mutants. We also found that the Rng1 mutant protein, which is truncated in its C-terminal domain encompassing HSR2, exhibited a residual processing activity against the 16S rRNA precursor, when overproduced. These results indicate that the HSR2 of RNase G plays an important role in substrate recognition and/or ribonucleolytic action.
Assuntos
Álcool Desidrogenase/genética , Aldeído Oxirredutases/genética , Endorribonucleases/genética , Endorribonucleases/metabolismo , Proteínas de Escherichia coli , Escherichia coli/enzimologia , Escherichia coli/genética , Complexos Multienzimáticos/genética , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , Substituição de Aminoácidos , Sequência de Bases , Mapeamento Cromossômico , Escherichia coli/metabolismo , Genes Bacterianos , Teste de Complementação Genética , Mutação Puntual , Precursores de RNA/genética , Precursores de RNA/metabolismo , Processamento Pós-Transcricional do RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Ribossômico 16S/genética , RNA Ribossômico 16S/metabolismoRESUMO
BACKGROUND: Escherichia coli rng gene (previously called cafA) encodes a novel RNase, named RNase G, which is involved in the 5' end-processing of 16S rRNA. In rng mutant cells, a precursor form of 16S rRNA, 16.3S rRNA, is accumulated. Here we report a role of RNase G in the in vivo mRNA metabolism. RESULTS: We found that rng:cat mutant strains overproduced a protein of about 100 kDa. N-terminal amino acid sequencing of this protein showed that it was identical to the fermentative alcohol dehydrogenase, the product of the adhE gene located at 28 min on the E. coli genetic map. The level of adhE mRNA was significantly higher in the rng:cat mutant strain than that in its parental strain, while such differences were not seen in other genes we examined. A rifampicin-chase experiment revealed that the half-life of adhE mRNA was 2.5-fold longer in the rng:cat disruptant than in the wild-type. CONCLUSION: These results indicate that, in addition to rRNA processing, RNase G is involved in in vivo mRNA degradation in E. coli.
Assuntos
Álcool Desidrogenase/metabolismo , Endorribonucleases/genética , Proteínas de Escherichia coli , Escherichia coli/metabolismo , RNA Mensageiro/metabolismo , RNA Ribossômico 16S/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Álcool Desidrogenase/genética , Sequência de Aminoácidos , Eletroforese em Gel de Poliacrilamida , Endorribonucleases/metabolismo , Escherichia coli/enzimologia , Escherichia coli/genética , Óperon Lac , Mutação , Filogenia , Reação em Cadeia da Polimerase , Precursores de RNA/genética , Processamento Pós-Transcricional do RNA , RNA Bacteriano/genética , RNA Ribossômico 16S/genética , Proteínas Recombinantes de Fusão/genética , Rifampina/metabolismoRESUMO
We found that the Escherichia coli cafA::cat mutant accumulated a precursor of 16S rRNA. This precursor migrated to the same position with 16.3S precursor found in the BUMMER strain that is known to be deficient in the 5' end processing of 16S rRNA. Accumulation of 16. 3S rRNA in the BUMMER mutant was complemented by introduction of a plasmid carrying the cafA gene. The mutant type cafA gene cloned from the BUMMER strain had a 11-bp deletion in its coding region. A small amount of the mature 16S rRNA was still formed in the cafA::cat mutant. This residual activity was found to be due to RNase E encoded by the rne/ams gene by rifampicin-chase experiments of the cafA::cat ams1 double mutant. These results indicated that the cafA gene encodes a novel RNase responsible for processing of the 5' end of 16S rRNA.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Escherichia coli , Escherichia coli/genética , Genes Bacterianos , RNA Ribossômico 16S/genética , Ribonucleases/genética , Clonagem Molecular , Endorribonucleases/metabolismo , Teste de Complementação Genética , Mutação , Plasmídeos , Precursores de RNA/genética , Processamento Pós-Transcricional do RNA , RNA Bacteriano/genética , Rifampina/metabolismoRESUMO
We analyzed the functional relationship between the Escherichia coli RNase E and the CafA protein, which show extensive sequence similarity. The temperature-sensitive growth of the RNase E mutant strain ams1 was partially suppressed by multicopy plasmids bearing the cafA gene. Introduction of a cafA::cat mutation enhanced the temperature sensitivity of the ams1 mutant. These results suggest that there is a functional homology between these two proteins.