Substrate structure requirements of the Pac1 ribonuclease from Schizosaccharmyces pombe.
The Pac1 ribonuclease of Schizosaccharomyces pombe is a member of the RNase III family of double-strand-specific ribonucleases. To examine RNA structural features required for efficient cleavage by the Pac1 RNase, we tested a variety of double-stranded and hairpin RNAs as substrates for the enzyme. The Pac1 RNase required substrates that have a minimal helix length of about 20 base pairs. The enzyme cut both strands of the helix at sites separated by two base pairs. However, Pac1 was also able to make a single-stranded cleavage within an internal bulge of an authentic Escherichia coli substrate at the same site chosen by RNase III. Pac1 efficiently degraded the structurally complex adenovirus VA RNA(I), but was inactive against the short HIV-1 TAR RNA hairpin. These results indicate that the Pac1 RNase prefers straight, perfect helices, but it can tolerate internal bulges that do not distort the helix severely. Like its homologue from Saccharomyces cerevisiae, the Pac1 RNase cleaved at two in vivo RNA processing sites in a hairpin structure in the 3' external transcribed spacer of the S. pombe pre-rRNA, suggesting a role for the enzyme in rRNA maturation.
Rotondo
G
G
Department of Microbiology, New York University School of Medicine, New York 10016, USA.
Huang
J Y
JY
Frendewey
D
D
D001483:Base Sequence; D002855:Chromatography, Thin Layer; D004722:Endoribonucleases / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins; D005656:Fungal Proteins; D016325:HIV Long Terminal Repeat / Q000235:genetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012322:RNA Precursors / Q000378:metabolism; D012330:RNA, Double-Stranded / Q000737:chemistry* / Q000378:metabolism*; D012342:RNA, Small Nuclear / Q000378:metabolism; D012367:RNA, Viral / Q000737:chemistry / Q000378:metabolism; D043244:Ribonuclease III; D029701:Saccharomyces cerevisiae Proteins; D012568:Schizosaccharomyces / Q000201:enzymology*; D029702:Schizosaccharomyces pombe Proteins; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D029968:Escherichia coli Proteins; D005656:Fungal Proteins; D012322:RNA Precursors; D012330:RNA, Double-Stranded; D012342:RNA, Small Nuclear; D012367:RNA, Viral; D029701:Saccharomyces cerevisiae Proteins; D029702:Schizosaccharomyces pombe Proteins; D004722:Endoribonucleases; C499540:pac1 protein, S pombe; C468367:RNT1 protein, S cerevisiae; D043244:Ribonuclease III; C468366:ribonuclease III, E coli