RNase footprinting of protein binding sites on an mRNA target of small RNAs.
Endoribonuclease footprinting is an important technique for probing RNA-protein interactions with single nucleotide resolution. The susceptibility of RNA residues to enzymatic digestion gives information about the RNA secondary structure, the location of protein binding sites, and the effects of protein binding on the RNA structure. Here we present a detailed protocol for using RNase T2, which cleaves single stranded RNA with a preference for A nucleotides, to footprint the protein Hfq on the rpoS mRNA leader. This protocol covers how to form the RNP complex, determine the correct dose of enzyme, footprint the protein, and analyze the cleavage pattern using primer extension.
T. C. Jenkins Department of Biophysics and CMDB Program, Johns Hopkins University, Baltimore, MD, USA.
Yi
Y
Peng
Toby J
TJ
Soper
Sarah A
SA
Woodson
D001426:Bacterial Proteins / Q000378:metabolism; D001665:Binding Sites; D017931:DNA Primers / Q000096:biosynthesis / Q000235:genetics; D004722:Endoribonucleases / Q000378:metabolism*; D035001:Host Factor 1 Protein / Q000378:metabolism*; D023302:Protein Footprinting / Q000379:methods*; D012329:RNA, Bacterial / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D058727:RNA, Small Untranslated / Q000378:metabolism*; D017421:Sequence Analysis; D012808:Sigma Factor / Q000378:metabolism
D001426:Bacterial Proteins; D017931:DNA Primers; D035001:Host Factor 1 Protein; D012329:RNA, Bacterial; D012333:RNA, Messenger; D058727:RNA, Small Untranslated; D012808:Sigma Factor; C069567:sigma factor KatF protein, Bacteria; D004722:Endoribonucleases; C022543:ribonuclease T(2)