Abstract
Transfer RNA nucleotidyltransferases (CCA-adding enzymes) are responsible for the maturation or repair of the functional 3′ end of tRNAs by means of the addition of the essential nucleotides CCA. However, it is unclear how tRNA nucleotidyltransferases polymerize CCA onto the 3′ terminus of immature tRNAs without using a nucleic acid template. Here we describe the crystal structure of the Archaeoglobus fulgidus tRNA nucleotidyltransferase in complex with tRNA. We also present ternary complexes of this enzyme with both RNA duplex mimics of the tRNA acceptor stem that terminate with the nucleotides C74 or C75, as well as the appropriate incoming nucleoside 5′-triphosphates. A single nucleotide-binding pocket exists whose specificity for both CTP and ATP is determined by the protein side chain of Arg 224 and backbone phosphates of the tRNA, which are non-complementary to and thus exclude UTP and GTP. Discrimination between CTP or ATP at a given addition step and at termination arises from changes in the size and shape of the nucleotide binding site that is progressively altered by the elongating 3′ end of the tRNA.
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Acknowledgements
We thank the beamline staff at X25 and X12C of the National Synchrotron Light Source, 8BM at the Advanced Photon Source, A1 at the Cornell High Energy Synchrotron Source, and 8.2.1 and 8.2.2 at the Advanced Light Source for assistance in data collection. We thank A. M. Weiner for suggestions on the manuscript; J. Wang for discussions; members of the Steitz laboratory for assistance at various stages of the project; and R. Evans for work with the ACC75 complex. This work was supported by an NIH grant.
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Supplementary information
Supplementary Figure S1
Regions of the 6.5 Å resolution electron density map for the AfCCA-tRNA complex calculated using 2Fo-Fc as coefficients and phases improved by solvent flattening and non-crystallographic symmetry averaging. (JPG 106 kb)
Supplementary Figure S2
Electron density from solvent-flattened and NCS-averaged 2Fo-Fc maps. (JPG 85 kb)
Supplementary Figure S3
The metal ion geometries in the AC74 (a) and ACC75 (b) complexes. (JPG 87 kb)
Supplementary Movie 1
Two tRNA molecules bound to the AfCCA dimer. (MP4 369 kb)
Supplementary Movie 2
The process of the addition of the last two nucleotides by the CCA-adding enzyme. (MP4 1999 kb)
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Xiong, Y., Steitz, T. Mechanism of transfer RNA maturation by CCA-adding enzyme without using an oligonucleotide template. Nature 430, 640–645 (2004). https://doi.org/10.1038/nature02711
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DOI: https://doi.org/10.1038/nature02711
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