RESUMO
The interactions of numerous regulatory small RNAs (sRNAs) with target mRNAs have been characterized, but how sRNAs can regulate multiple, structurally unrelated mRNAs is less understood. Here we show that Salmonella GcvB sRNA directly acts on seven target mRNAs that commonly encode periplasmic substrate-binding proteins of ABC uptake systems for amino acids and peptides. Alignment of GcvB homologs of distantly related bacteria revealed a conserved G/U-rich element that is strictly required for GcvB target recognition. Analysis of target gene fusion regulation in vivo, and in vitro structure probing and translation assays showed that GcvB represses its target mRNAs by binding to extended C/A-rich regions, which may also serve as translational enhancer elements. In some cases (oppA, dppA), GcvB repression can be explained by masking the ribosome-binding site (RBS) to prevent 30S subunit binding. However, GcvB can also effectively repress translation by binding to target mRNAs at upstream sites, outside the RBS. Specifically, GcvB represses gltI mRNA translation at the C/A-rich target site located at positions -57 to -45 relative to the start codon. Taken together, our study suggests highly conserved regions in sRNAs and mRNA regions distant from Shine-Dalgarno sequences as important elements for the identification of sRNA targets.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , MicroRNAs/fisiologia , RNA Mensageiro/metabolismo , Ribossomos/metabolismo , Salmonella/genética , Proteínas de Bactérias/genética , Composição de Bases , Sequência de Bases , Sítios de Ligação , Regulação Bacteriana da Expressão Gênica , MicroRNAs/metabolismo , Modelos Biológicos , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Proteínas Periplásmicas de Ligação/genética , Biossíntese de Proteínas/genética , RNA Mensageiro/química , Elementos Reguladores de Transcrição/fisiologia , Homologia de Sequência do Ácido NucleicoRESUMO
Binding-protein-dependent secondary transporters make up a unique transport protein family. They use a solute-binding protein in proton-motive-force-driven transport. Only a few systems have been functionally analysed. The yiaMNO genes of Escherichia coli K-12 encode one family member that transports the rare pentose l-xylulose. Its physiological role is unknown, since wild-type E. coli K-12 does not utilize l-xylulose as sole carbon source. Deletion of the yiaMNO genes in E. coli K-12 strain MC4100 resulted in remarkable changes in the transition from exponential growth to the stationary phase, high-salt survival and biofilm formation.
Assuntos
Proteínas de Transporte/metabolismo , Escherichia coli K12/crescimento & desenvolvimento , Deleção de Genes , Regulação Bacteriana da Expressão Gênica , Proteínas de Membrana Transportadoras/metabolismo , Biofilmes/crescimento & desenvolvimento , Transporte Biológico , Proteínas de Transporte/genética , Meios de Cultura , Escherichia coli K12/efeitos dos fármacos , Escherichia coli K12/genética , Escherichia coli K12/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Membrana Transportadoras/genética , Óperon , Transdução de Sinais , Cloreto de Sódio/farmacologia , Xilulose/metabolismoRESUMO
The yiaQRS genes of Escherichia coli K-12 are involved in carbohydrate metabolism. Clustering of homologous genes was found throughout several unrelated bacteria. Strikingly, all four bacterial transport protein classes were found, conserving transport function but not mechanism. It appears that during evolution the ability to transport, phosphorylate and metabolize substrates of unknown identity have been conserved. However, the transporter classes have been swapped. This probably demonstrates the subtlety of transport-protein evolution.
Assuntos
Bactérias/genética , Metabolismo dos Carboidratos , Evolução Molecular , Proteínas de Membrana Transportadoras/genética , Família Multigênica , Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Transporte Biológico , Proteínas de Membrana Transportadoras/metabolismoRESUMO
The yiaMNO genes of Escherichia coli K-12 encode a binding protein-dependent secondary, or tri-partite ATP-independent periplasmic (TRAP), transporter. Since only a few members of this family have been functionally characterized to date, we aimed to identify the substrate for this transporter. Cells that constitutively express the yiaK-S gene cluster metabolized the rare pentose L-xylulose, while deletion of the yiaMNO transporter genes reduced L-xylulose metabolism. The periplasmic substrate-binding protein YiaO was found to bind L-xylulose, and stimulated L-xylulose uptake by spheroplasts. These date indicate that the yiaMNO transporter mediates uptake of this rare pentose.
