Proteolysis of Saccharomyces cerevisiae RNase H1 in E coli.
Expression of S cerevisiae RNase H1 in E coli leads to the formation of a proteolytic product with a molecular mass of 30 kDa that is derived from the 39-kDa full length protein. The 30-kDa form retains RNase H1 activity, as determined by renaturation gel assay. The amount of proteolysis observed depends on the procedure used in preparing the cell extracts for protein analysis. The cleavage site on the amino acid sequence of the 39-kDa RNase H1 was determined by N-terminal sequence analysis of the 30-kDa proteolytic form. The cut occurs between two arginines located at the amino terminus region of the protein. The pattern of proteolysis was examined for both the wild-type RNase H1 and a mutant RNase H1 that was constructed in this work. In the mutant the second arginine of the cleavage site was changed to a lysine. Comparisons of the expression of the wild-type and altered protein in two different E coli strains demonstrate that the protease responsible for the degradation has a specificity very similar to that of the OmpT protease. However, the proteolysis observed in an OmpT background in extracts, prepared by boiling the cells in SDS containing buffer, indicates that the protease may, unlike OH108.
Cerritelli
S M
SM
Laboratory of Molecular Genetics and Endocrinology and Reproduction, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892.
Shin
D Y
DY
Chen
H C
HC
Gonzales
M
M
Crouch
R J
RJ
D000595:Amino Acid Sequence; D004926:Escherichia coli / Q000378:metabolism*; D008970:Molecular Weight; D009154:Mutation; D010447:Peptide Hydrolases / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D016914:Ribonuclease H / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*
D011994:Recombinant Proteins; D016914:Ribonuclease H; D010447:Peptide Hydrolases