Determination of the catalytic parameters of the N-terminal half of Escherichia coli ribonuclease E and the identification of critical functional groups in RNA substrates.
Ribonuclease E is required for the rapid decay and correct processing of RNA in Escherichia coli. A detailed understanding of the hydrolysis of RNA by this and related enzymes will require the integration of structural and molecular data with quantitative measurements of RNA hydrolysis. Therefore, an assay for RNaseE that can be set up to have relatively high throughput while being sensitive and quantitative will be advantageous. Here we describe such an assay, which is based on the automated high pressure liquid chromatography analysis of fluorescently labeled RNA samples. We have used this assay to optimize reaction conditions, to determine for the first time the catalytic parameters for a polypeptide of RNaseE, and to investigate the RNaseE-catalyzed reaction through the modification of functional groups within an RNA substrate. We find that catalysis is dependent on both protonated and unprotonated functional groups and that the recognition of a guanosine sequence determinant that is upstream of the scissile bond appears to consist of interactions with the exocyclic 2-amino group, the 7N of the nucleobase and the imino proton or 6-keto group. Additionally, we find that a ribose-like sugar conformation is preferred in the 5'-nucleotide of the scissile phosphodiester bond and that a 2'-hydroxyl group proton is not essential. Steric bulk at the 2' position in the 5'-nucleotide appears to be inhibitory to the reaction. Combined, these observations establish a foundation for the functional interpretation of a three-dimensional structure of the catalytic domain of RNaseE when solved.
Redko
Yulia
Y
Astbury Centre for Structural Molecular Biology, Faculty of Biological Sciences, University of Leeds, Manton Building, LS2 9JT Leeds, United Kingdom.
Tock
Mark R
MR
Adams
Chris J
CJ
Kaberdin
Vladimir R
VR
Grasby
Jane A
JA
McDowall
Kenneth J
KJ
D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D004722:Endoribonucleases / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D005456:Fluorescent Dyes; D006151:Guanosine / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000378:metabolism; D012313:RNA / Q000737:chemistry* / Q000378:metabolism*; D012965:Sodium Chloride / Q000494:pharmacology; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D005456:Fluorescent Dyes; D009841:Oligonucleotides; D006151:Guanosine; D012965:Sodium Chloride; D012313:RNA; D004722:Endoribonucleases; C022537:ribonuclease E; D008274:Magnesium