NIH Research Festival
The bacterial Hfq is an RNA-binding chaperone that stabilizes small noncoding RNAs (sRNA) and facilitates riboregulation by base pairing of the sRNA with target mRNAs. The Hfq has a conserved N-terminal core, which is sufficient to bind RNA and promote pairing to targets, and a less conserved long C-terminal. Previous reports provided conflicting assessments of the necessity for the C-terminus, possibly reflecting different approaches and assays used by different groups. We constructed strains of E. coli in which Hfq deleted for the C-terminus (hfq65) is expressed from the chromosomal locus, allowing in vivo comparisons with the wild-type Hfq, expressed under similar conditions and at physiological levels. Class I sRNA binds proximal and rim, whereas Class II interact with proximal and distal faces of Hfq. We compared Class I and Class II sRNA accumulation by northern blot analysis in wild-type Hfq and hfq65 strains. Class II but not most Class I sRNAs had reduced accumulation in the hfq65 strain compared to wild-type. One tested Class II sRNA (ChiX) was also defective for function in the hfq65 host. We found that the C-terminus did not affect intrinsic stability or binding of Class II sRNAs to Hfq. Rather, in the absence of the Hfq C-terminus, Class II sRNAs are more rapidly released and degraded after pairing with sRNAs. These results, coupled with in vitro studies of truncated Hfq carried out by our collaborators are consistent with a model in which the Hfq C-terminus helps to protect the protein from non-specific nucleic acid binding.
Scientific Focus Area: Molecular Biology and Biochemistry
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