A method for in vivo identification of bacterial small RNA-binding proteins.
Bottom Line: Subsequently, PrrF- and PrrH-protein complexes were enriched using cDNA "bait", and enriched RNA-protein complexes were analyzed by tandem mass spectrometry to identify PrrF and PrrH associated proteins.Interestingly, Hfq was identified more often in samples probed with the PrrF cDNA "bait" as compared to the PrrH cDNA "bait", suggesting Hfq has a stronger binding affinity for the PrrF sRNAs in vivo.As such, this study demonstrates that in vivo cross-linking coupled with sequence-specific affinity chromatography and tandem mass spectrometry (SSAC-MS/MS) is an effective methodology for unbiased identification of bacterial sRNA-binding proteins.
Affiliation: Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, Baltimore, Maryland.Show MeSH
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Mentions: To determine the validity of our SSAC-MS/MS results and further examine the interaction of Hfq with the PrrF and PrrH sRNAs, we cloned, over-expressed, and purified the P. aeruginosa Hfq protein as described in the Materials and Methods (Fig. 3A and B). The identity of the purified Hfq protein shown in Figure 3B was confirmed by mass spectrometry and used for EMSA's with a mixture of the PrrF and PrrH sRNAs, generated by in vitro transcription of the entire prrF locus. Hfq-sRNA-binding reactions were resolved by native gel electrophoresis and analyzed for mobility of the PrrF and PrrH sRNAs. These results showed a shift in mobility of the PrrF sRNAs in the presence of 44 ng/μL of Hfq, corresponding to nearly a 50-fold molar ratio of Hfq to the PrrF and PrrH sRNA mixture (Fig. 3C). In contrast, shifts in PrrH mobility were detected at Hfq concentrations as low as 11 ng/μL, corresponding to approximately a 12-to-1 molar ratio of Hfq and the PrrF and PrrH sRNAs, (Fig. 3C). The shifted PrrF and PrrH bands were eliminated by the addition of 20-fold excess of unlabeled PrrF and PrrH sRNA (Fig. 3D), indicating these bands correspond to a specific interaction of the PrrF and PrrH sRNAs with Hfq. Thus, our data suggest that Hfq has a somewhat higher affinity for the PrrH sRNA as compared to PrrF under these experimental conditions.
Affiliation: Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, Baltimore, Maryland.