On the recognition of helical RNA by cobra venom V1 nuclease.
The V1 nuclease from cobra venom preferentially hydrolyzes double helical RNA and has been used extensively for detecting RNA secondary structure. To increase the utility of this enzyme as an RNA structure probe, we have investigated its properties and substrate specificity, using assays for polynucleotide hydrolysis based on fluorescent polynucleotide derivatives. Enzymatic activity requires both Na+ and Mg2+, with optima at 100 and 0.3 mM, respectively. From the sharp decrease in enzyme activity above 100 mM Na+ we estimate that 3-4 ionic interactions between the protein and polynucleotide phosphates take place. Analysis of products remaining after extensive V1 digestion also shows that the minimum size substrate is 4-6 nucleotides long. Helical RNAs and DNAs have Michaelis constants a factor of 3-10 times lower than most single-stranded RNAs. However, poly(epsilon A) has a Michaelis constant equal to the best synthetic double helices tested and is hydrolyzed at a rate comparable to helical RNA. The major V1 cutting sites in yeast tRNAPhe have Michaelis constants lower than any synthetic polymers. These data suggest that V1 nuclease recognizes any 4-6-nucleotide segment of polynucleotide backbone with an approximately helical conformation, but does not require that the bases be paired in a helix. A few single-stranded V1 cleavage sites are known in tRNA and rRNA, and their structures are consistent with the suggested V1 recognition site.
H B
HB
Lowman
D E
DE
Draper
D001483:Base Sequence; D004247:DNA / Q000378:metabolism; D004722:Endoribonucleases / Q000378:metabolism*; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D009690:Nucleic Acid Conformation; D011119:Polynucleotides / Q000378:metabolism; D012313:RNA / Q000378:metabolism*; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism; D012964:Sodium / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity
D011119:Polynucleotides; D012346:RNA, Transfer, Amino Acyl; D012313:RNA; D004247:DNA; D012964:Sodium; D004722:Endoribonucleases; C032825:ribonuclease V(1); D008274:Magnesium