Interactions of the RNA-Binding Protein Hfq with cspA mRNA, Encoding the Major Cold Shock Protein

Abstract

ABSTRACT 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 ∼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.