RNA sequence and secondary structure participate in high-affinity CsrA-RNA interaction

被引:215
作者
Dubey, AK
Baker, CS
Romeo, T
Babitzke, P [1 ]
机构
[1] Penn State Univ, Dept Biochem & Mol Biol, University Pk, PA 16802 USA
[2] Emory Univ, Sch Med, Dept Microbiol & Immunol, Atlanta, GA 30322 USA
关键词
RNA binding protein; translation control; protein-RNA interaction; SELEX;
D O I
10.1261/rna.2990205
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The global Csr regulatory system controls bacterial gene expression post-transcriptionally. CsrA of Escherichia coli is an RNA binding protein that plays a central role in repressing several stationary phase processes and activating certain exponential phase functions. CsrA regulates translation initiation of several genes by binding to the mRNA leaders and blocking ribosome binding. CsrB and CsrC are noncoding regulatory RNAs that are capable of sequestering CsrA and antagonizing its activity. Each of the known target transcripts contains multiple CsrA binding sites, although considerable sequence variation exists among these RNA targets, with GGA being the most highly conserved element. High-affinity RNA ligands containing single CsrA binding sites were identified from a combinatorial library using systematic evolution of ligands by exponential enrichment (SELEX). The SELEX-derived consensus was determined as RUACARGGAUGU, with the ACA and GGA motifs being 100% conserved and the GU sequence being present in all but one ligand. The majority (51/55) of the RNAs contained GGA in the loop of a hairpin within the most stable predicted structure, an arrangement similar to several natural CsrA binding sites. Strikingly, the identity of several nucleotides that were predicted to form base pairs in each stem were 100% conserved, suggesting that primary sequence information was embedded within the base-paired region. The affinity of CsrA for several selected ligands was measured using quantitative gel mobility shift assays. A mutational analysis of one selected ligand confirmed that the conserved ACA, GGA, and GU residues were critical for CsrA binding and that RNA secondary structure participates in CsrA-RNA recognition.
引用
收藏
页码:1579 / 1587
页数:9
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