Probing the substrate specificity of Escherichia coli RNase E using a novel oligonucleotide-based assay.
Endoribonuclease RNase E has a central role in both processing and decay of RNA in Escherichia coli, and apparently in many other organisms, where RNase E homologs were identified or their existence has been predicted from genomic data. Although the biochemical properties of this enzyme have been already studied for many years, the substrate specificity of RNase E is still poorly characterized. Here, I have described a novel oligonucleotide-based assay to identify specific sequence determinants that either facilitate or impede the recognition and cleavage of RNA by the catalytic domain of the enzyme. The knowledge of these determinants is crucial for understanding the nature of RNA-protein interactions that control the specificity and efficiency of RNase E cleavage and opens new perspectives for further studies of this multi-domain protein. Moreover, the simplicity and efficiency of the proposed assay suggest that it can be a valuable tool not only for the characterization of RNase E homologs but also for the analysis of other site-specific nucleases.
Kaberdin
Vladimir R
VR
Max F. Perutz Laboratories, Department of Microbiology and Genetics, University Department at the Vienna Biocenter, Dr. Bohrgasse 9/4, A-1030 Vienna, Austria. vladimir@gem.univie.ac.at
D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D004722:Endoribonucleases / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D009154:Mutation; D009841:Oligonucleotides / Q000235:genetics / Q000378:metabolism*; D011061:Poly A / Q000235:genetics / Q000378:metabolism; D011072:Poly U / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
D009841:Oligonucleotides; D011061:Poly A; D011072:Poly U; D004722:Endoribonucleases; C022537:ribonuclease E