RESUMO
SR1 is a dual-function sRNA that acts as a base-pairing regulatory RNA on the ahrC mRNA and as a peptide-encoding mRNA on the gapA operon. The SR1-encoded peptide SR1P binds GapA thereby stabilizing gapA mRNA. Under glycolytic conditions, SR1 transcription is repressed by CcpN and CcpA. A computer-based search identified 23 SR1 homologues in Bacillus, Geobacillus, Anoxybacillus and Brevibacillus species. All homologues share a high structural identity with Bacillus subtilis SR1, and the encoded SR1P peptides are highly similar. In the Bacillus cereus group, the sr1p region is present in triplicate or duplicate resulting in longer SR1 species. In all cases, sr1 expression is under control of CcpN, and transcriptional lacZ fusions of nine examined SR1 homologues were sensitive to glucose. Two homologues showed an additional glucose-independent repression by CcpN and an unknown factor. A total of 10 out of 11 tested SR1P homologues complemented a B. subtilis Δsr1 strain in their ability to stabilize gapA mRNA, but only five of them bound GapA tightly. In vitro binding assays with six SR1/ahrC pairs suggest that-despite divergent primary sequences-the base-pairing function is also preserved. In summary, SR1 is an sRNA with two functions that have been conserved over ≈1 billion years.
Assuntos
Bacillus subtilis/genética , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , Pequeno RNA não Traduzido/genética , Pequeno RNA não Traduzido/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Cisteína/fisiologia , Regulação Bacteriana da Expressão Gênica , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Peptídeos/química , Filogenia , Regiões Promotoras Genéticas , Estabilidade de RNA , RNA Bacteriano/química , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Pequeno RNA não Traduzido/química , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Sintenia , Transativadores/genéticaRESUMO
Here, we describe bsrG/SR4, a novel type I toxin-antitoxin system from the SPß prophage region of the Bacillus subtilis chromosome. The 294-nucleotide bsrG RNA encodes a 38-amino-acid toxin, whereas SR4 is a 180-nucleotide antisense RNA that acts as the antitoxin. Both genes overlap by 123 nucleotides. BsrG expression increases at the onset of stationary phase. The sr4 promoter is 6- to 10-fold stronger than the bsrG promoter. Deletion of sr4 stabilizes bsrG mRNA and causes cell lysis on agar plates, which is due to the BsrG peptide and not the bsrG mRNA. SR4 overexpression could compensate cell lysis caused by overexpression of bsrG. SR4 interacts with the 3' UTR of bsrG RNA, thereby promoting its degradation. RNase III cleaves the bsrG RNA/SR4 duplex at position 185 of bsrG RNA, but is not essential for the function of the toxin-antitoxin system. Endoribonuclease Y and 3'-5' exoribonuclease R participate in the degradation of both bsrG RNA and SR4, whereas PnpA processes three SR4 precursors to the mature RNA. A heat shock at 48°C results in faster degradation and, therefore, significantly decreased amounts of bsrG RNA.
Assuntos
Bacillus subtilis/genética , Toxinas Bacterianas/genética , RNA Antissenso/genética , Sequência de Bases , Mapeamento Cromossômico , Cromossomos Bacterianos/genética , DNA Bacteriano/genética , Regulação Bacteriana da Expressão Gênica , Técnicas de Inativação de Genes , Dados de Sequência Molecular , Regiões Promotoras Genéticas , RNA Bacteriano/genética , Ribonuclease III/metabolismo , TemperaturaRESUMO
Regulatory small RNAs (sRNAs) in bacterial genomes have become a focus of research over the past 8 years. Whereas more than 100 such sRNAs have been found in Escherichia coli, relatively little is known about sRNAs in gram-positive bacteria. Using a computational approach, we identified two sRNAs in intergenic regions of the Bacillus subtilis genome, SR1 and SR2 (renamed BsrF). Recently, we demonstrated that SR1 inhibits the translation initiation of the transcriptional activator AhrC. Here, we describe detection of BsrF, its expression profile, and its regulation by CodY. Furthermore, we mapped the secondary structure of BsrF. BsrF is expressed in complex and minimal media in all growth phases in B. subtilis and, with a similar expression profile, also in Bacillus amyloliquefaciens. Neither overexpression nor deletion of bsrF affected the growth of B. subtilis. BsrF was found to be long-lived in complex and minimal media. Analysis of 13 putative transcription factor binding sites upstream of bsrF revealed only an effect for CodY. Here, we showed by using Northern blotting, lacZ reporter gene fusions, in vitro transcription, and DNase I footprinting that the transcription of bsrF is activated by CodY in the presence of branched-chain amino acids and GTP. Furthermore, BsrF transcription was increased 1.5- to 2-fold by glucose in the presence of branched-chain amino acids, and this increase was independent of the known glucose-dependent regulators. BsrF is the second target for which transcriptional activation by CodY has been discovered.