Biochemical and genomic analysis of substrate recognition by the double-stranded RNA binding domain of yeast RNase III.
Members of the RNase III family of double-stranded RNA (dsRNA) endonucleases are important enzymes of RNA metabolism in eukaryotic cells. Rnt1p is the only known member of the RNase III family of endonucleases in Saccharomyces cerevisiae. Previous studies have shown that Rnt1p cleaves dsRNA capped by a conserved AGNN tetraloop motif, which is a major determinant for Rnt1p binding and cleavage. The solution structure of the dsRNA-binding domain (dsRBD) of Rnt1p bound to a cognate RNA substrate revealed the structural basis for binding of the conserved tetraloop motif by alpha-helix 1 of the dsRBD. In this study, we have analyzed extensively the effects of mutations of helix 1 residues that contact the RNA. We show, using microarray analysis, that mutations of these amino acids induce substrate-specific processing defects in vivo. Cleavage kinetics and binding studies show that these mutations affect RNA cleavage and binding in vitro to different extents and suggest a function for some specific amino acids of the dsRBD in the catalytic positioning of the enzyme. Moreover, we show that 2'-hydroxyl groups of nucleotides of the tetraloop or adjacent base pairs predicted to interact with residues of alpha-helix 1 are important for Rnt1p cleavage in vitro. This study underscores the importance of a few amino acid contacts for positioning of a dsRBD onto its RNA target, and implicates the specific orientation of helix 1 on the RNA for proper positioning of the catalytic domain.
Department of Chemistry and Biochemistry, University of California, Los Angeles, CA 90095-1569, USA.
Anthony K
AK
Henras
Mui
M
Sam
Shawna L
SL
Hiley
Haihong
H
Wu
Timothy R
TR
Hughes
Juli
J
Feigon
Guillaume F
GF
Chanfreau
D001483:Base Sequence; D001665:Binding Sites; D007700:Kinetics; D008715:Methionine; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D011487:Protein Conformation; D012330:RNA, Double-Stranded / Q000737:chemistry / Q000378:metabolism*; D012331:RNA, Fungal / Q000737:chemistry / Q000378:metabolism; D043244:Ribonuclease III / Q000737:chemistry / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity
D012330:RNA, Double-Stranded; D012331:RNA, Fungal; D029701:Saccharomyces cerevisiae Proteins; D008715:Methionine; C468367:RNT1 protein, S cerevisiae; D043244:Ribonuclease III