Global maps of ProQ binding in vivo reveal target recognition via RNA structure and stability control at mRNA 3' ends. Global maps of ProQ binding in vivo reveal target recognition via RNA structure and stability control at mRNA 3' ends

In this study binding sites for the RNA-binding protein ProQ was determined in Salmonella and Escherichia coli Overall design: To detect the binding sites for Salmonella or E. coli ProQ we collected three biological replicates of Salmonella strain SL1344 and two biological replicates of E. coli strain MG1655 where the proQ gene was fused to a 3xFLAG tag. Half of each replicate culture was irradiated with UV light (254 nm, 800 mJ/cm2) (+CL), while the other half was left untreated (-CL). Bacteria were lysed and the FLAG-tagged protein was immunoprecipitated using a monoclonal anti-FLAG antibody. The samples were treated with benzonase nuclease, calf intestine phophorylase, and polynucleotide kinase in the presence of radioactive gamma-ATP. Samples were separated with SDS-PAGE followed by transfer to nitrocellulose membranes. Radioactively labelled RNA-protein complexes were eluted from membranes and treated with Proteinase K. Purified RNA was used as input for library preparation using the NEB Next Small RNA Library kit.