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
Mentions: Northern analysis of the PrrF and PrrH sRNAs was performed as previously described with some modifications (Oglesby-Sherrouse and Vasil 2010). Briefly, 10–20 μg of total RNA isolated on RNeasy Mini Columns was run on a 6% polyacrylamide denaturing (7 mol/L urea) gel then transferred to a BrightStar membrane (Life Technologies, Grand Island, NY, USA) using a semi-dry transfer apparatus. Biotinylated oligonucleotides that were complementary to the regions of PrrF1, PrrF2, or PrrH as shown in Figure 1A were purchased from Integrated DNA Technologies (IDT) and hybridized to blots overnight at 42°C. The membrane was washed using the Northern Max Low Stringency and High Stringency wash solutions according to the manufacturer's instructions. Detection of the biotinylated probes was carried out using the BrightStar BioDetect nonisotopic detection kit (Life Technologies).
Affiliation: Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, Baltimore, Maryland.