CLONING, SEQUENCE-ANALYSIS, OVERPRODUCTION IN ESCHERICHIA-COLI AND ENZYMATIC CHARACTERIZATION OF THE RNASE HI FROM MYCOBACTERIUM-SMEGMATIS

Activity gel analysis of cell extracts from slow- and fast-growing myc obacteria confirmed the presence of several RNase H activities in both classes of organism. The rnhA gene from Mycobacterium smegmatis (Ms) was subsequently cloned using an internal gene segment probe [Mizrahi et al., Gene 136 (1993) 287-290]. The gene encodes a polypeptide of 15 9 amino acids that shares 50% identity with the RNase HI from Escheric hia coli (Ec). However, unlike its counterparts from Gram(-) bacteria, Ms rnhA does not form an overlapping divergent transcriptional unit w ith dnaQ (encoding the epsilon (proofreading) subunit of DNA polymeras e III), Ms RNase HI was overproduced in Ec as an enzymatically active maltose-binding protein (MBP) fusion protein which cleaved the RNA str and of an RNA . DNA hybrid with a similar site selectivity to that of its Ec homologue.