Sequence-selective interactions with RNA by CspB, CspC and CspE, members of the CspA family of Escherichia coli.
The CspA family of Escherichia coli comprises nine homologous proteins, CspA to CspI. CspA, the major cold shock protein, binds RNA with low sequence specificity and low binding affinity. This is considered to be important for its proposed function as an RNA chaperone to prevent the formation of secondary structures in RNA molecules, thus facilitating translation at low temperature. The cellular functions of other Csp proteins are yet to be fully elucidated, and their sequence specific binding capabilities have not been identified. As a step towards identification of the target genes of Csp proteins, we investigated the RNA binding specificities of CspB, CspC and CspE by an in vitro selection approach (SELEX). In the present study, we show that these proteins are able to bind preferentially to specific RNA/single-stranded DNA sequences. The consensus sequences for CspB, CspC and CspE are U/T stretches, AGGGAGGGA and AU/AT-rich regions, especially AAAUUU, respectively. CspE and CspB have Kd values in the range 0.23-0.9 x 10(-6) M, while CspC has 10-fold lower binding affinity. Consistent with our recent findings of transcriptional regulation of cspA by CspE, we have identified a motif identical to the CspE consensus. This motif is the putative CspE-mediated transcription pause recognition site in a 5'-untranslated region of the cspA mRNA.
Phadtare
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Department of Biochemistry, Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, NJ 08854, USA.
Inouye
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D001426:Bacterial Proteins; D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D002352:Carrier Proteins / Q000378:metabolism*; D017124:Conserved Sequence; D004247:DNA / Q000378:metabolism; D004269:DNA, Bacterial / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins; D005784:Gene Amplification; D005821:Genetic Techniques; D006360:Heat-Shock Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D012329:RNA, Bacterial / Q000378:metabolism*; D016601:RNA-Binding Proteins; D012091:Repetitive Sequences, Nucleic Acid; D012641:Selection, Genetic; D013379:Substrate Specificity
D001426:Bacterial Proteins; D002352:Carrier Proteins; C491179:CspB protein, E coli; C088099:CspC protein, bacteria; C088097:CspE protein, E coli; D004269:DNA, Bacterial; D004277:DNA, Single-Stranded; D029968:Escherichia coli Proteins; D006360:Heat-Shock Proteins; D012329:RNA, Bacterial; D016601:RNA-Binding Proteins; C077188:cold-shock protein CspB, Bacteria; C490241:cspC protein, E coli; D004247:DNA