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.[Keywords: Small RNA; riboregulator; post-transcriptional control; translation inhibition; ABC transporter; GcvB] Supplemental material is available at http://www.genesdev.org.
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.
The transporter associated with antigen processing (TAP) is essential for peptide loading onto major histocompatibility (MHC) class I molecules by translocating peptides into the endoplasmic reticulum. We have explored the conditions for detergent solubilization of functionally active, heterologously expressed human TAP from microsomal membranes. The efficiency to solubilize TAP was tested for a variety of detergents as well as for different solubilization conditions. The activity of the solubilized TAP complex was analyzed over time, using a non-radioactive crosslinking assay with a photo-activateable peptide, in the presence or absence of external lipid. The detergent CHAPS was found optimally to retain activity and thus allowed us to reconstitute detergent-solubilized, active TAP into proteoliposomes.
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.
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.
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