Probing the catalytic triad of an archaeal RNA splicing endonuclease.
Among the four known mechanisms of intron removal, three are reputedly catalyzed by RNA molecules. In the fourth mechanism, a protein endonuclease removes introns from nuclear tRNA and all archaeal RNAs. Three strictly conserved residues of the splicing endonuclease, a histidine, a lysine, and a tyrosine, were predicted to catalyze the intron cleavage reaction in a manner similar to that of the catalytic triad of ribonuclease A. Single-turnover kinetic parameters were obtained for the wild-type enzyme and two triad mutants. Mutation of histidine to alanine produced an at least approximately 28-fold reduction; mutation of tyrosine to phenylalanine produced an at least approximately 7-fold reduction in activity, while a histidine and tyrosine double mutation abolished cleavage. The single mutation of lysine to glutamic acid abolished RNA cleavage activity in the absence of a divalent metal but maintained a substantial level of activity in the presence of specific divalent metals. These data support important functional roles already proposed for the catalytic triad and suggest an intriguing hypothesis in which the splicing endonuclease is an intermediate in the transition from the RNA to the RNP world.
Calvin
Kate
K
Department of Chemistry and Biochemistry, Institute of Molecular Biophysics, Florida State University, Tallahassee, Florida 32306, USA.
Xue
Song
S
Ellis
Charles
C
Mitchell
Michelle H
MH
Li
Hong
H
D001665:Binding Sites; D002384:Catalysis; D017931:DNA Primers / Q000737:chemistry; D004926:Escherichia coli / Q000378:metabolism; D019143:Evolution, Molecular; D018698:Glutamic Acid / Q000737:chemistry; D007438:Introns; D008239:Lysine / Q000737:chemistry; D008968:Molecular Conformation; D009154:Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012313:RNA / Q000737:chemistry; D019642:RNA, Archaeal / Q000235:genetics*; D020124:Thermotoga maritima / Q000378:metabolism
D017931:DNA Primers; D019642:RNA, Archaeal; D018698:Glutamic Acid; D012313:RNA; D008239:Lysine