Identification of the Hfq-binding site on DsrA RNA: Hfq binds without altering DsrA secondary structure.
DsrA RNA regulates the translation of two global regulatory proteins in Escherichia coli. DsrA activates the translation of RpoS while repressing the translation of H-NS. The RNA-binding protein Hfq is necessary for DsrA to function in vivo. Although Hfq binds to DsrA in vitro, the role of Hfq in DsrA-mediated regulation is not known. One hypothesis was that Hfq acts as an RNA chaperone by unfolding DsrA, thereby facilitating interactions with target RNAs. To test this hypothesis, we have examined the structure of DsrA bound to Hfq in vitro. Comparison of free DsrA to DsrA bound to Hfq by RNase footprinting, circular dichroism, and thermal melt profiles shows that Hfq does not alter DsrA secondary structures, but might affect its tertiary conformation. We identify the site on DsrA where Hfq binds, which is a structural element in the middle of DsrA. In addition, we show that although long poly(U) RNAs compete with DsrA for binding to Hfq, a short poly(U) stretch present in DsrA is not necessary for Hfq binding. Finally, unlike other RNAs, DsrA binding to Hfq is not competed with by poly(A) RNA. In fact, DsrA:poly(A):Hfq may form a stable ternary complex, raising the possibility that Hfq has multiple RNA-binding sites.
Brescia
Cristin C
CC
Department of Microbiology and Immunology, Medical College of Ohio, Toledo 43614, USA.
Mikulecky
Peter J
PJ
Feig
Andrew L
AL
Sledjeski
Darren D
DD
D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000235:genetics*; D001483:Base Sequence; D001665:Binding Sites; D002942:Circular Dichroism; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D035001:Host Factor 1 Protein / Q000378:metabolism*; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D012329:RNA, Bacterial / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D001425:Bacterial Outer Membrane Proteins; C499660:DrsA protein, E coli; D029968:Escherichia coli Proteins; D035001:Host Factor 1 Protein; D012329:RNA, Bacterial