title: On the role of a conserved, potentially helix-breaking residue in the tRNA-binding alpha-helix of archaeal CCA-adding enzymes.
abstract: Archaeal class I CCA-adding enzymes use a ribonucleoprotein template to build and repair the universally conserved 3'-terminal CCA sequence of the acceptor stem of all tRNAs. A wealth of structural and biochemical data indicate that the Archaeoglobus fulgidus CCA-adding enzyme binds primarily to the tRNA acceptor stem through a long, highly conserved alpha-helix that lies nearly parallel to the acceptor stem and makes many contacts with its sugar-phosphate backbone. Although the geometry of this alpha-helix is nearly ideal in all available cocrystal structures, the helix contains a highly conserved, potentially helix-breaking proline or glycine near the N terminus. We performed a mutational analysis to dissect the role of this residue in CCA-addition activity. We found that the phylogenetically permissible P295G mutant and the phylogenetically absent P295T had little effect on CCA addition, whereas P295A and P295S progressively interfered with CCA addition (C74>C75>A76 addition). We also examined the effects of these mutations on tRNA binding and the kinetics of CCA addition, and performed a computational analysis using Rosetta Design to better understand the role of P295 in nucleotide transfer. Our data indicate that CCA-adding activity does not correlate with the stability of the pre-addition cocrystal structures visualized by X-ray crystallography. Rather, the data are consistent with a transient conformational change involving P295 of the tRNA-binding alpha-helix during or between one or more steps in CCA addition.
authors.affiliation: Department of Biochemistry, School of Medicine, University of Washington, Seattle, Washington 98195-7350, USA.
authors.forename: Hyundae D
authors.initials: HD
authors.lastname: Cho
authors.forename: Vanita D
authors.initials: VD
authors.lastname: Sood
authors.forename: David
authors.initials: D
authors.lastname: Baker
authors.forename: Alan M
authors.initials: AM
authors.lastname: Weiner
mesh_terms: D019943:Amino Acid Substitution; D019611:Archaeoglobus fulgidus / Q000201:enzymology / Q000378:metabolism*; D003198:Computer Simulation; D018360:Crystallography, X-Ray; D008958:Models, Molecular; D016296:Mutagenesis; D009690:Nucleic Acid Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012316:RNA Nucleotidyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012343:RNA, Transfer / Q000378:metabolism*; D012984:Software
chemical_list: D012343:RNA, Transfer; D012316:RNA Nucleotidyltransferases; C023198:tRNA nucleotidyltransferase