Probing of tertiary interactions in RNA: 2'-hydroxyl-base contacts between the RNase P RNA and pre-tRNA.
A general method has been developed to analyze all 2' hydroxyl groups involved in tertiary interactions in RNA in a single experiment. This method involves comparing the activity of populations of circularly permuted RNAs that contain or lack potential hydrogen-bond donors at each position. The 2' hydroxyls of the pre-tRNA substrate identified as potential hydrogen bond donors in intermolecular interactions with the ribozyme from eubacterial RNase P (P RNA) are located in the T stem and T loop, acceptor stem, and 3' CCA regions. To locate the hydrogen-bond acceptors for one of those 2' hydroxyls in the P RNA, a phylogenetically conserved adenosine was mutated to a guanosine. When this mutant P RNA was used, increased cleavage activity of a single circularly permuted substrate within the population was observed. The cleavage efficiency (kcat/Km) of a singly 2'-deoxy-substituted substrate at this position in the T stem was also determined. For the wild-type P RNA, the catalytic efficiency was significantly decreased compared with that of the all-ribo substrate, consistent with the notion that this 2' hydroxyl plays an important role. For the P RNA mutant, no additional effect was found upon 2'-deoxy substitution. We propose that this particular 2' hydroxyl in the pre-tRNA interacts specifically with this adenosine in the P RNA. This method should be useful in examining the role of 2' hydroxyl groups in other RNA-RNA and RNA-protein complexes.
Pan
T
T
Department of Biochemistry and Molecular Biology, University of Chicago, IL 60637, USA.
Loria
A
A
Zhong
K
K
D000241:Adenosine; D001665:Binding Sites; D004722:Endoribonucleases / Q000737:chemistry* / Q000378:metabolism*; D005051:Eubacterium / Q000201:enzymology; D006860:Hydrogen Bonding; D008961:Models, Structural; D009690:Nucleic Acid Conformation; D017354:Point Mutation; D012322:RNA Precursors / Q000737:chemistry* / Q000378:metabolism*; D012329:RNA, Bacterial / Q000737:chemistry / Q000378:metabolism; D016337:RNA, Catalytic / Q000737:chemistry* / Q000378:metabolism*; D012360:RNA, Transfer, Phe / Q000737:chemistry* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D043262:Ribonuclease P; D013379:Substrate Specificity
D012322:RNA Precursors; D012329:RNA, Bacterial; D016337:RNA, Catalytic; D012360:RNA, Transfer, Phe; D011994:Recombinant Proteins; D004722:Endoribonucleases; D043262:Ribonuclease P; D000241:Adenosine