Interaction of the Neurospora crassa mitochondrial tyrosyl-tRNA synthetase (CYT-18 protein) with the group I intron P4-P6 domain. Thermodynamic analysis and the role of metal ions.
The Neurospora crassa mitochondrial tyrosyl-tRNA synthetase (CYT-18 protein) functions in splicing group I introns by promoting the formation of the catalytically active structure of the intron's catalytic core. Previous studies suggested a model in which the protein binds first to the intron's P4-P6 domain, and then makes additional contacts with the P3-P9 domain to stabilize the two domains in the correct relative orientation to form the intron's active site. Here, we analyzed the interaction of CYT-18 with a small RNA (P4-P6 RNA) corresponding to the isolated P4-P6 domain of the N. crassa mitochondrial large subunit ribosomal RNA intron. RNA footprinting and modification-interference experiments showed that CYT-18 binds to this small RNA around the junction of the P4-P6 stacked helices on the side opposite the active-site cleft, as it does to the P4-P6 domain in the intact intron. The binding is inhibited by chemical modifications that disrupt base-pairing in P4, P6, and P6a, indicating that a partially folded structure of the P4-P6 domain is required. The temperature-dependence of binding indicates that the interaction is driven by a favorable enthalpy change, but is accompanied by an unfavorable entropy change. The latter may reflect entropically unfavorable conformational changes or decreased conformational flexibility in the complex. CYT-18 binding is inhibited at > or =125 mM KCl, indicating a strong dependence on phosphodiester-backbone interactions. On the other hand, Mg(2+) is absolutely required for CYT-18 binding, with titration experiments showing approximately 1.5 magnesium ions bound per complex. Metal ion-cleavage experiments identified a divalent cation-binding site near the boundary of P6 and J6/6a, and chemical modification showed that Mg(2+) binding induces RNA conformational changes in this region, as well as elsewhere, particularly in J4/5. Together, these findings suggest a model in which the binding of Mg(2+) near J6/6a and possibly at one additional location in the P4-P6 RNA induces formation of a specific phosphodiester-backbone geometry that is required for CYT-18 binding. The binding of CYT-18 may then establish the correct structure at the junction of the P4/P6 stacked helices for assembly of the P3-P9 domain. The interaction of CYT-18 with the P4-P6 domain appears similar to the TyrRS interaction with the D-/anticodon arm stacked helices of tRNA(Tyr).
Caprara
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MG
Institute for Cellular and Molecular Biology, Department of Chemistry and Biochemistry, Section of Molecular Genetics and Microbiology, School of Biological Sciences, University of Texas at Austin, Austin, TX 78712, USA.
Myers
C A
CA
Lambowitz
A M
AM
D001483:Base Sequence; D002412:Cations / Q000378:metabolism / Q000494:pharmacology; D004047:Diethyl Pyrocarbonate / Q000378:metabolism; D019277:Entropy; D005038:Ethylnitrosourea / Q000378:metabolism; D007438:Introns / Q000235:genetics*; D007455:Iodine / Q000378:metabolism; D008274:Magnesium / Q000378:metabolism / Q000494:pharmacology; D008670:Metals / Q000378:metabolism* / Q000494:pharmacology; D008928:Mitochondria / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D009492:Neurospora crassa / Q000201:enzymology* / Q000235:genetics; D009690:Nucleic Acid Conformation / Q000187:drug effects; D018583:Pliability; D011189:Potassium Chloride / Q000378:metabolism / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects; D012331:RNA, Fungal / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012365:RNA, Transfer, Tyr / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000378:metabolism; D013463:Sulfuric Acid Esters / Q000378:metabolism; D013696:Temperature; D014448:Tyrosine-tRNA Ligase / Q000378:metabolism*
D002412:Cations; D008670:Metals; D012331:RNA, Fungal; D012365:RNA, Transfer, Tyr; D016601:RNA-Binding Proteins; D013463:Sulfuric Acid Esters; D011189:Potassium Chloride; D007455:Iodine; D014448:Tyrosine-tRNA Ligase; D008274:Magnesium; C007482:dimethyl sulfate; D004047:Diethyl Pyrocarbonate; D005038:Ethylnitrosourea