Characterization of the rnc-97 mutation of RNAaseIII: a glycine to glutamate substitution increases the requirement for magnesium ions.
The rnc-97 mutation of the Escherichia coli double-stranded-RNA-specific ribonuclease III (RNAaseIII) was previously isolated by virtue of the lethal expression of RNAaseIII in Saccharomyces cerevisiae. Here we show that rnc-97 is a single point mutation causing the substitution of glycine 97 by glutamic acid. The mutation eliminates the lethal phenotype of RNAaseIII expression in yeast and reduces fourfold the effect of RNAaseIII expression on bacteriophage gy1 propagation in E. coli. Mutant RNAaseIII-G97E and wild-type RNAaseIII were purified according to published procedures. The apparent molecular masses of the two enzymes on SDS polyacrylamide gels are the same but they differ in pI (6.85 for RNAaseIII-G97E and 7.3 for RNAaseIII). Whereas the two enzymes (under standard assay conditions) do not show a great difference in activity towards double-stranded RNA and defined single-stranded RNAaseIII substrates, they differ dramatically (20-fold or more) under conditions of Mg2+ limitation. The hypothesis that limitation of Mg2+ ions in vivo is responsible for the phenotypes of the rnc-97 mutation in S. cerevisiae and E. coli is discussed.
Department of Molecular Biology, Hebrew University-Hadassah Medical School, Jerusalem, Israel.
Y
Y
Davidov
A
A
Rahat
I
I
Flechner
O
O
Pines
D004722:Endoribonucleases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D029968:Escherichia coli Proteins; D007526:Isoelectric Point; D008274:Magnesium / Q000378:metabolism*; D008970:Molecular Weight; D010641:Phenotype; D017354:Point Mutation; D043244:Ribonuclease III; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D017422:Sequence Analysis, DNA
D029968:Escherichia coli Proteins; D029701:Saccharomyces cerevisiae Proteins; D004722:Endoribonucleases; C468367:RNT1 protein, S cerevisiae; D043244:Ribonuclease III; C468366:ribonuclease III, E coli; D008274:Magnesium