Proteolysis of Saccharomyces cerevisiae RNase H1 in E coli》.pdf

Biochimie (1993) 75, 107-111 107 © Soci6t6 franqaise de biochimie et biologie mol6culaire / Elsevier, Paris Proteolysis of Saccharomyces cerevisiae RNase HI in E coil S M Cerritellia, D Y Shina, H C Chenb, M Gonzales, RJ Crouch~, aLaboratory of Molecular Genetics and IEndocrinology and Reproduction Research Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA (Received 29 September; accepted 20 November 1992) Summary ~ Expression of S cerevisiae RNase H I 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 HI activity, as detennined by renaturation gel assay. The amount o~ 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 H! 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 HI and a mutant RNase HI 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 diffe- rent E coli strains demonstrate th