Analysis of the CYT-18 Protein Binding Site at the Junction of Stacked Helices in a Group I Intron RNA by Quantitative Binding Assays andin vitroSelection |
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| Affiliation: | 1. Department of Biophysics, Johns Hopkins University, 3400 N. Charles St., MD 21218, USA;2. Department of Chemistry, College of Chemistry and Chemical Engineering, and Key Laboratory for Chemical Biology of Fujian Province, Xiamen University, Xiamen, Fujian 361005, China;3. Institute of Drug Discovery Technology, Ningbo University, Ningbo, Zhejiang 315211, China;1. Department of Microbiology, University of Pennsylvania, Philadelphia, PA 19104, USA;1. Laboratory of Medicinal Chemistry, Research Institute of Pharmaceutical Sciences, College of Pharmacy, Seoul National University, Seoul 08826, Republic of Korea;2. Medicinal Bioconvergence Research Center, College of Pharmacy, Seoul National University, Seoul 08826, Republic of Korea;3. Department of Molecular Medicine and Biopharmaceutical Sciences, Graduate School of Convergence Science and Technology, Seoul National University, Seoul 08826, Republic of Korea;4. Department of Global Medical Science, Sungshin University, Seoul 01133, Republic of Korea;5. Department of Pharmaceutical Chemistry, Hanoi University of Pharmacy, Hanoi, Viet Nam |
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| Abstract: | TheNeurospora crassamitochondrial tyrosyl-tRNA synthetase (CYT-18 protein) functions in splicing group I introns by promoting the formation of the catalytically active structure of the intron RNA. Previous studies showed that CYT-18 binds with high affinity to the P4-P6 domain of the catalytic core and that there is some additional contribution to binding from the P3-P9 domain. Here, quantitative binding assays with deletion derivatives of theN. crassamitochondrial large rRNA intron showed that at least 70% of the binding energy can be accounted for by the interaction of CYT-18 with the P4-P6 domain. Within this domain, P4 and P6 are required for high affinity CYT-18 binding, while the distal elements P5 and P6a may contribute indirectly by stabilizing the correct structure of the binding site in P4 and P6. CYT-18 binds to a small RNA corresponding to the isolated P4-P6 domain, but not to a permuted version of this RNA in which P4-P6 is a continuous rather than a stacked helix. Iterativein vitroselection experiments with the isolated P4-P6 domain showed a requirement for base-pairing to maintain helices P4, P6 and P6a, but indicate that P5 is subject to fewer constraints. The most strongly conserved nucleotides in the selections were clustered around the junction of the P4-P6 stacked helix, with ten nucleotides (J3/4-2,3, P4 bp -1 and 3, and P6 bp -1 and 2) found invariant in the context of the wild-type RNA structure.In vitromutagenesis confirmed that replacement of the wild-type nucleotides at J3/4-2 and 3 or P4 bp-3 markedly decreased CYT-18 binding, reflecting either base specific contacts or indirect readout of RNA structure by the protein. Our results suggest that a major function of CYT-18 is to promote assembly of the P4-P6 domain by stabilizing the correct geometry at the junction of the P4-P6 stacked helix. The relatively large number of conserved nucleotides at the binding site suggests that the interaction of CYT-18 with group I introns is unlikely to have arisen by chance and could reflect either an evolutionary relationship between group I introns and tRNAs or interaction with a common stacked-helical structural motif that evolved separately in these RNAs. |
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