RESUMO
BACKGROUND: Small, untranslated RNA molecules were identified initially in bacteria, but examples can be found in all kingdoms of life. These RNAs carry out diverse functions, and many of them are regulators of gene expression. Genes encoding small, untranslated RNAs are difficult to detect experimentally or to predict by traditional sequence analysis approaches. Thus, in spite of the rising recognition that such RNAs may play key roles in bacterial physiology, many of the small RNAs known to date were discovered fortuitously. RESULTS: To search the Escherichia coli genome sequence for genes encoding small RNAs, we developed a computational strategy employing transcription signals and genomic features of the known small RNA-encoding genes. The search, for which we used rather restrictive criteria, has led to the prediction of 24 putative sRNA-encoding genes, of which 23 were tested experimentally. Here we report on the discovery of 14 genes encoding novel small RNAs in E. coli and their expression patterns under a variety of physiological conditions. Most of the newly discovered RNAs are abundant. Interestingly, the expression level of a significant number of these RNAs increases upon entry into stationary phase. CONCLUSIONS: Based on our results, we conclude that small RNAs are much more widespread than previously imagined and that these versatile molecules may play important roles in the fine-tuning of cell responses to changing environments.
Assuntos
DNA Intergênico , Escherichia coli/genética , RNA não Traduzido/genética , Transcrição Gênica , Northern Blotting , Mapeamento Cromossômico , Oligodesoxirribonucleotídeos/genética , Oligodesoxirribonucleotídeos/metabolismo , Regiões Promotoras Genéticas/genética , RNA Bacteriano/genética , RNA não Traduzido/metabolismoRESUMO
OxyS is a small untranslated RNA that is induced in response to oxidative stress in Escherichia coli. This small RNA acts as a global regulator affecting the expression of multiple genes. OxyS represses the translation of fhlA, a transcriptional activator for formate metabolism. Previously, we have shown that fhlA repression by OxyS is mediated through base-pairing with a short sequence overlapping the ribosome binding site. Here we show that the OxyS-fhlA interaction involves a second site residing further downstream, within the coding region of fhlA. Mutations that disrupt pairing at this site affect the ability of OxyS to prevent 30 S ribosomes from binding to fhlA mRNA. Structure probing of fhlA mRNA demonstrates that both sites reside in the loops of two stem-loop structures. OxyS-fhlA pairing analysis shows that OxyS binds wild-type fhlA with an apparent dissociation constant of 25 nM, indicating that kissing complex formation between OxyS and fhlA results in a stable antisense-target complex. Mutations at either site, which disrupt pairing of OxyS to fhlA, decrease the stability of this complex. Our results indicate that kissing complex formation is sufficient to repress fhlA translation by OxyS.
Assuntos
Proteínas de Escherichia coli , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica/genética , Estresse Oxidativo/genética , RNA Antissenso/genética , RNA Bacteriano/genética , Transativadores/genética , Sequência de Bases , Sítios de Ligação , Escherichia coli/metabolismo , Genes Reporter/genética , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Conformação de Ácido Nucleico , Hibridização de Ácido Nucleico , Biossíntese de Proteínas/genética , Estabilidade de RNA , RNA Antissenso/química , RNA Antissenso/metabolismo , RNA Bacteriano/química , RNA Bacteriano/metabolismo , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sequências Reguladoras de Ácido Nucleico/genética , Ribossomos/química , Ribossomos/genética , Ribossomos/metabolismo , Ésteres do Ácido Sulfúrico/metabolismo , TermodinâmicaRESUMO
The OxyS regulatory RNA integrates the adaptive response to hydrogen peroxide with other cellular stress responses and protects against DNA damage. Among the OxyS targets is the rpoS-encoded sigma(s) subunit of RNA polymerase. Sigma(s) is a central regulator of genes induced by osmotic stress, starvation and entry into stationary phase. We examined the mechanism whereby OxyS represses rpoS expression and found that the OxyS RNA inhibits translation of the rpoS message. This repression is dependent on the hfq-encoded RNA-binding protein (also denoted host factor I, HF-I). Co-immunoprecipitation and gel mobility shift experiments revealed that the OxyS RNA binds Hfq, suggesting that OxyS represses rpoS translation by altering Hfq activity.
Assuntos
Proteínas de Bactérias/fisiologia , Proteínas de Transporte/fisiologia , Proteínas de Ligação a DNA , Proteínas de Escherichia coli , RNA Bacteriano/fisiologia , Sequências Reguladoras de Ácido Nucleico/fisiologia , Proteínas Repressoras/fisiologia , Fator sigma/fisiologia , Regiões não Traduzidas/fisiologia , Sequência de Bases , Escherichia coli , Fator Proteico 1 do Hospedeiro , Fatores Hospedeiros de Integração , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Estresse Oxidativo/fisiologia , Testes de Precipitina , Proteínas de Ligação a RNA/fisiologia , Fatores de Transcrição/fisiologiaRESUMO
OxyS is a small untranslated RNA which is induced in response to oxidative stress in Escherichia coli. This novel RNA acts as a global regulator to activate or repress the expression of as many as 40 genes, including the fhlA-encoded transcriptional activator and the rpoS-encoded sigma(s) subunit of RNA polymerase. Deletion analysis of OxyS showed that different domains of the small RNA are required for the regulation of fhlA and rpoS. We examined the mechanism of OxyS repression of fhlA and found that the OxyS RNA inhibits fhlA translation by pairing with a short sequence overlapping the Shine-Dalgarno sequence, thereby blocking ribosome binding/translation.
