RESUMO
The Escherichia coli stationary phase transcription factor RpoS is translated in response to small noncoding RNAs (sRNAs), which base pair with the rpoS mRNA leader. The bacterial Sm-like protein Hfq anneals sRNAs with their mRNA targets by simultaneously binding the mRNA and sRNA. Intriguingly, Hfq is recruited to the rpoS leader via AAN motifs far upstream of the sRNA. SHAPE (selective 2'-hydroxyl acylation and primer extension) chemical footprinting showed that the rpoS leader is divided into a far upstream domain, an Hfq binding domain, and a downstream inhibitory stem-loop containing the sRNA and ribosome binding sites. To investigate how Hfq promotes sRNA-mRNA base pairing from a distance, we deleted the natural AAN Hfq binding site, and we inserted artificial AAN binding sites at various positions in the rpoS leader. All the relocated AAN motifs restored tight Hfq binding in vitro, but only insertion at the natural position restored Hfq-dependent sRNA annealing in vitro and sRNA regulation of rpoS translation in vivo. Furthermore, U-rich motifs in the downstream inhibitory domain stabilized the rpoS mRNA-Hfq complex and contributed to regulation of rpoS expression. We propose that the natural Hfq binding domain is optimal for positive regulation because it recruits Hfq to the mRNA and allows it to act on incoming sRNAs without opening the inhibitory stem-loop when sRNA is absent.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Regulação Bacteriana da Expressão Gênica , Fator Proteico 1 do Hospedeiro/metabolismo , RNA Mensageiro/metabolismo , Pequeno RNA não Traduzido/metabolismo , Fator sigma/genética , Regiões 5' não Traduzidas , Conformação de Ácido Nucleico , Ligação Proteica , Biossíntese de Proteínas , RNA Mensageiro/genética , Pequeno RNA não Traduzido/genéticaRESUMO
Endoribonuclease footprinting is an important technique for probing RNA-protein interactions with single nucleotide resolution. The susceptibility of RNA residues to enzymatic digestion gives information about the RNA secondary structure, the location of protein binding sites, and the effects of protein binding on the RNA structure. Here we present a detailed protocol for using RNase T2, which cleaves single stranded RNA with a preference for A nucleotides, to footprint the protein Hfq on the rpoS mRNA leader. This protocol covers how to form the RNP complex, determine the correct dose of enzyme, footprint the protein, and analyze the cleavage pattern using primer extension.
Assuntos
Endorribonucleases/metabolismo , Fator Proteico 1 do Hospedeiro/metabolismo , Pegadas de Proteínas/métodos , RNA Bacteriano/metabolismo , Pequeno RNA não Traduzido/metabolismo , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Primers do DNA/biossíntese , Primers do DNA/genética , RNA Mensageiro/metabolismo , Análise de Sequência , Fator sigma/metabolismoRESUMO
Bacterial small RNAs (sRNAs) modulate gene expression by base-pairing with target mRNAs. Many sRNAs require the Sm-like RNA binding protein Hfq as a cofactor. Well-characterized interactions between DsrA sRNA and the rpoS mRNA leader were used to understand how Hfq stimulates sRNA pairing with target mRNAs. DsrA annealing stimulates expression of rpoS by disrupting a secondary structure in the rpoS leader, which otherwise prevents translation. Both RNAs bind Hfq with similar affinity but interact with opposite faces of the Hfq hexamer. Using mutations that block interactions between two of the three components, we demonstrate that Hfq binding to a functionally critical (AAN)(4) motif in rpoS mRNA rescues DsrA binding to a hyperstable rpoS mutant. We also show that Hfq cannot stably bridge the RNAs. Persistent ternary complexes only form when the two RNAs are complementary. Thus, Hfq mainly acts by binding and restructuring the rpoS mRNA. However, Hfq binding to DsrA is needed for maximum annealing in vitro, indicating that transient interactions with both RNAs contribute to the regulatory mechanism.
Assuntos
Fator Proteico 1 do Hospedeiro/metabolismo , RNA Bacteriano/metabolismo , RNA Mensageiro/metabolismo , RNA não Traduzido/metabolismo , Staphylococcus aureus/metabolismo , Fator Proteico 1 do Hospedeiro/química , Modelos Moleculares , Mutação , Conformação de Ácido Nucleico , Ligação Proteica , Estrutura Terciária de Proteína , RNA Bacteriano/química , RNA Bacteriano/genética , RNA Mensageiro/química , RNA Mensageiro/genética , Pequeno RNA não Traduzido , RNA não Traduzido/química , RNA não Traduzido/genética , Staphylococcus aureus/químicaRESUMO
Small noncoding RNAs (sRNAs) regulate the response of bacteria to environmental stress in conjunction with the Sm-like RNA binding protein Hfq. DsrA sRNA stimulates translation of the RpoS stress response factor in Escherichia coli by base-pairing with the 5' leader of the rpoS mRNA and opening a stem-loop that represses translation initiation. We report that rpoS leader sequences upstream of this stem-loop greatly increase the sensitivity of rpoS mRNA to Hfq and DsrA. Native gel mobility shift assays show that Hfq increases the rate of DsrA binding to the full 576 nt rpoS leader as much as 50-fold. By contrast, base-pairing with a 138-nt RNA containing just the repressor stem-loop is accelerated only twofold. Deletion and mutagenesis experiments showed that sensitivity to Hfq requires an upstream AAYAA sequence. Leaders long enough to contain this sequence bind Hfq tightly and form stable ternary complexes with Hfq and DsrA. A model is proposed in which Hfq recruits DsrA to the rpoS mRNA by binding both RNAs, releasing the self-repressing structure in the mRNA. Once base-pairing between DsrA and rpoS mRNA is established, interactions between Hfq and the mRNA may stabilize the RNA complex by removing Hfq from the sRNA.