The track of the pre-tRNA 5' leader in the ribonuclease P ribozyme-substrate complex.
The ribonuclease P (RNase P) ribozyme is an endonuclease that binds precursor tRNAs and catalyzes the removal of 5' leader nucleotides. Biochemical and photo-cross-linking studies have identified sites of contact between the mature tRNA domain of pre-tRNA and the ribozyme; however, relatively little is known about the location of the 5' leader in the ribozyme-substrate complex. To investigate the local three-dimensional environment of the 5' leader, we employed the short-range photo-cross-linking agent 4-thiouridine (s(4)U). The s(4)U photoagent was incorporated into a series of pre-tRNA substrates containing unique uridine residues in the 5' leader sequence at positions -1, -3, -5, -7, or -10. The modified substrates formed high-affinity complexes with the ribozyme and produced discrete intermolecular cross-links to RNase P RNA from Bacillus subtilis. Locations of the cross-linked nucleotides in the ribozyme and pre-tRNA were determined by reverse transcriptase primer extension. Photoagents incorporated into the 5' leader detected discrete elements of ribozyme structure in a progression from J18/2 to L15 to P3. Importantly, all of the cross-linked species retained the ability to cleave the covalently attached pre-tRNA, indicating that the cross-links reflect the native structure of the ribozyme-substrate complex. Together with available structural and biochemical data, the cross-linking results suggest a model for the position of the 5' leader within the ground-state ribozyme-substrate complex.
Christian
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Center for RNA Molecular Biology, Department of Molecular Biology and Microbiology, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106, USA.
Harris
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D001483:Base Sequence; D004722:Endoribonucleases / Q000378:metabolism*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012322:RNA Precursors / Q000737:chemistry / Q000378:metabolism*; D016337:RNA, Catalytic / Q000378:metabolism*; D012343:RNA, Transfer / Q000737:chemistry / Q000378:metabolism*; D043262:Ribonuclease P; D013379:Substrate Specificity
D012322:RNA Precursors; D016337:RNA, Catalytic; D012343:RNA, Transfer; D004722:Endoribonucleases; D043262:Ribonuclease P