Interactions of the RNA-binding protein Hfq with cspA mRNA, encoding the major cold shock protein.
CspA, a small protein that is highly induced by cold shock, is encoded by a monocistronic mRNA of 428 nucleotides (nt) whose half-life and abundance are greatly increased following cold shock. We show here that in vitro cspA mRNA can bind multiple copies of Hfq, a hexameric Sm-like protein which promotes a variety of RNA-RNA interactions. Binding of the first Hfq hexamer occurs with an apparent K(d) (dissociation constant) of <40 nM; up to seven additional hexamers can bind sequentially at higher concentrations. Known ligands of Hfq, including the small regulatory RNA, RyhB, compete with cspA mRNA. Several experiments suggest that the first binding site to be occupied by Hfq is located at or near the 3' end of cspA mRNA. The consequences of limited Hfq binding in vitro include nearly total inhibition of RNase E cleavage at a site approximately 35 nt from the 3' end of the mRNA, stimulation of polyadenylation by poly(A) polymerase 1, and subsequent exonucleolytic degradation by polynucleotide phosphorylase. We propose that Hfq may play a facilitating role in the metabolism of cspA mRNA.
Department of Biochemistry and Molecular Biology, The University of British Columbia, Vancouver, BC, Canada V6T 1Z3.
J S
JS
Hankins
H
H
Denroche
G A
GA
Mackie
D001483:Base Sequence; D001665:Binding Sites; D057773:Cold Shock Proteins and Peptides; D004722:Endoribonucleases / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000378:metabolism*; D006360:Heat-Shock Proteins / Q000235:genetics*; D035001:Host Factor 1 Protein / Q000378:metabolism*; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000378:metabolism; D026723:Polyadenylation; D011117:Polyribonucleotide Nucleotidyltransferase / Q000378:metabolism; D011485:Protein Binding; D020365:RNA Helicases / Q000378:metabolism; D020871:RNA Stability; D012333:RNA, Messenger / Q000378:metabolism*; D013696:Temperature
D057773:Cold Shock Proteins and Peptides; C498192:CspA protein, E coli; D029968:Escherichia coli Proteins; D006360:Heat-Shock Proteins; C087373:Hfq protein, E coli; D035001:Host Factor 1 Protein; D009097:Multienzyme Complexes; D012333:RNA, Messenger; C111244:degradosome; D011117:Polyribonucleotide Nucleotidyltransferase; D004722:Endoribonucleases; D020365:RNA Helicases