The cleavage specificity of RNase

We determined sites in X ell mRNA that are cleaved by RNase III in the presence of X OOP antisense RNA, using a series of OOP RNAs with different internal deletions. In OOP RNA-cll mRNA structures containing a potential region of continuous double-stranded RNA bounded by a non-complementary unpaired region, RNase III cleaved the ell mRNA at one or more preferred sites located 10 to 14 bases from the 3'-end of the region of continuous complementarity. Cleavage patterns were almost identical when the presumptive structure was the same continuously double-stranded region followed by a single-stranded bulge and a second short region of base pairing. The sequences of the new cleavage sites show generally good agreement with a consensus sequence derived from thirty-five previously determined cleavage sequences. In contrast, four 'non-sites' at which cleavage is never observed show poor agreement with this consensus sequence. We conclude that RNase III specificity is determined both by the distance from the end of continuous pairing and by nucleotide sequence features within the region of pairing. INTRODUCTION The Escherichia coli enzyme RNase III was first isolated on the basis of its specificity for double-stranded RNA in vitro (1). Closely spaced, often staggered single strand breaks at intervals of approximately 15 bases are produced by the enzyme using high-molecular-weight double-stranded RNA substrates (1-3) . Since the product molecules are of nearly uniform length it is argued that the enzyme exhibits little or no sequence specificity in acting on high-molecular-weight double-stranded RNA (4), and that the major determinant of a site of cleavage may be the distance from the end of the double-stranded molecule. Length, however, would not seem to be the sole determinant since the product molecules are not all of precisely the same length (2). A number of cellular functions have been discovered for RNase ITI, including processing of E. coli ribosomal RNA precursor molecules (5-8) , and processing the polycistronic transcript of the bacteriophage T7 early region (5,9,10). RNase El processing sites flank the bacteriophage T3 S-adenosylmethionine hydrolase gene (11), and are found in the E. coli rpUL-rpoBC operon (12), and the mRNA transcripts for the E. coli metf2 (13), and pnp and rpsO genes (14,15). The transcript coding for the E. coli RNase III gene also has a cleavage site for RNase in (16). The bacteriophage X int gene is regulated by RNase HI (17—21), and RNase III processing sites occur elsewhere on X transcripts (22—24). The complex of sar antisense RNA with ant mRNA in bacteriophage P22 is cleaved by RNase m (25). The RNA molecules cleaved by RNase IE all are capable of forming doublestranded regions in the range of 20 to 40 bp, often with a short internal unpaired region in the vicinity of the RNase HI cleavage sites. In these cases a limited homology among RNase Illsensitive sites has been detected (7). RNase III is in some cases responsible for gene regulation by antisense RNA, such as the regulation of X ell gene expression by X OOP antisense RNA (26,27), and the regulation of the E. coli copT gene by the cop A antisense RNA (28). OOP RNA is a 77 base transcript that can form a perfect double helix with its target mRNA molecule, a major rightward transcript of X that extends hundreds of nucleotides to either side of the complementary region. OOP RNA results in RNase HI cleavage in the target cII-0 mRNA at 13 nucleotides from the 3'-end of the region of complementarity with OOP RNA. When an OOP RNA with an internal deletion opposite this cleavage site is paired with wild-type cR-0 mRNA, cleavage no longer occurs at the normal site, and cleavage is detected instead at a new site 22 bases to the 5'-side of the initial cleavage site (27). This new cleavage site is located 13 bases from the end of a region of continuous complementarity between the deleted OOP RNA and cII-O mRNA. This raises the question of whether, in attacking a continuously double-stranded region of considerable length, RNase III always cleaves at precisely 13 nucleotides from the 3'-end. In this paper we determine the cleavage sites for antisense RNA molecules with internal deletions that extend progressively further into into the molecule. We show that cleavage does not always occur at precisely 13 nucleotides from the 3'-end of the complementary region, but rather occurs at one or more of several preferred sites located 10 to 14 nucleotides from the 3'-end of the complementary region. MATERIALS AND METHODS Bacteria and plasmids E. coli bacterial strains producing cU-0 mRNA are UC6171 (F~ galK~ hfl-l str~ sup°) (29) and its isogenic RNase III" derivative UC5993 (F~ galK' hfl-l mc\05 glyA:Xc6 str~ sup), * To whom correspondence should be addressed + Present address: Division of Biology, California Institute of Technology, Pasadena, CA 91125, USA 4810 Nucleic Acids Research, Vol. 18, No. 16 lysogenic for \int-6 clts857 croll Pantf (26). E. coli bacterial strains producing cU-cat mRNA are UC6650 and its isogenic RNase ni" derivative UC6659. The strain UC6650 is a derivative of UC6171 that has inserted into its galK gene a X DNA fragment containing the X temperature sensitive clts857 repressor gene and the X N gene under control of the strong repressible X p^ promoter followed by a kanR gene controlled by its own promoter, and inserted into its lacZ gene a fragment of DNA in which the X pL promoter governs expression of the X ell gene and a chloramphenicol transacetylase (cat) gene (Fig. 1). To insert the X dts857, X N, and kanR sequences into the E. coli galK gene, the desired DNA fragments were first assembled into a plasmid between two half galK gene DNA fragments. This plasmid (pMEMl 1) was introduced into a gal host and a Gal" derivative was isolated that had incorporated plasmid DNA into the host chromosome by homologous recombination. The inserted DNA was then transferred to strain UC6171 by PI transduction, selecting for kanamycin resistance. The resulting strain, UC6444, was Kan, Amp, immune to X at 32° and sensitive to X at 42°. The strain UC6444 grows normally at 42°. To insert the X pL, X cU and cat sequences into the lacZ of UC6444, the desired DNA fragments were first assembled into a plasmid between two half lacZ gene sequences that are missing small portions of the extreme 5'-end, 3'-end and middle regions of the gene. In this plasmid (pMEM49) the X pL and X ell gene fragments are bounded by unique HindHI and BamHI sites, allowing any Hindlll-BamHI DNA fragment to be inserted in their place. A Sail site is located between the cat and lacZ gene sequences, allowing the entire insert to be replaced by any HindlU-Sall DNA fragment. The DNA inserted into the lacZ sequences in the plasmid was introduced into the chromosome by homologous recombination, followed by elimination of the plasmid through growth at 42° and PI-mediated transduction to UC6444, selecting for Cm' resistance at 42°. The resulting strain is Amp and Lac~, and Cm at 42° and Cm at 32°. galK/2 dts857 PL galK/2