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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.

SUBMITTER: Cho HD

PROVIDER: S-EPMC2441987 | biostudies-literature | 2008 Jul

REPOSITORIES: biostudies-literature

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On the role of a conserved, potentially helix-breaking residue in the tRNA-binding alpha-helix of archaeal CCA-adding enzymes.

Cho Hyundae D HD Sood Vanita D VD Baker David D Weiner Alan M AM

RNA (New York, N.Y.) 20080521 7

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 ...[more]

PMID: 18495940

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Project description:RNase Z is a widely distributed and usually essential endoribonuclease involved in the 3'-end maturation of transfer RNAs (tRNAs). A CCA triplet that is needed for tRNA aminoacylation in protein translation is added by a nucleotidyl-transferase after the 3'-end processing by RNase Z. However, a considerable proportion of the archaeal pre-tRNAs genetically encode a CCA motif, while the enzymatic characteristics of the archaeal RNase (aRNase) Zs in processing CCA-containing pre-tRNAs remain unclear. This study intensively characterized two methanomicrobial aRNase Zs, the Methanolobus psychrophilus mpy-RNase Z and the Methanococcus maripaludis mmp-RNase Z, particularly focusing on the properties of processing the CCA-containing pre-tRNAs, and in parallel comparison with a bacterial bsu-RNase Z from Bacillus subtilis. Kinetic analysis found that Co2+ supplementation enhanced the cleavage efficiency of mpy-RNase Z, mmp-RNase Z, and bsu-RNase Z for 1400-, 2990-, and 34-fold, respectively, and Co2+ is even more indispensable to the aRNase Zs than to bsu-RNase Z. Mg2+ also elevated the initial cleavage velocity (V0) of bsu-RNase Z for 60.5-fold. The two aRNase Zs exhibited indiscriminate efficiencies in processing CCA-containing vs. CCA-less pre-tRNAs. However, V0 of bsu-RNase Z was markedly reduced for 1520-fold by the CCA motif present in pre-tRNAs under Mg2+ supplementation, but only 5.8-fold reduced under Co2+ supplementation, suggesting Co2+ could ameliorate the CCA motif inhibition on bsu-RNase Z. By 3'-RACE, we determined that the aRNase Zs cleaved just downstream the discriminator nucleotide and the CCA triplet in CCA-less and CCA-containing pre-tRNAs, thus exposing the 3'-end for linking CCA and the genetically encoded CCA triplet, respectively. The aRNase Zs, but not bsu-RNase Z, were also able to process the intron-embedded archaeal pre-tRNAs, and even process pre-tRNAs that lack the D, T, or anticodon arm, but strictly required the acceptor stem. In summary, the two methanomicrobial aRNase Zs use cobalt as a metal ligand and process a broad spectrum of pre-tRNAs, and the characteristics would extend our understandings on aRNase Zs.

Dataset Information

On the role of a conserved, potentially helix-breaking residue in the tRNA-binding alpha-helix of archaeal CCA-adding enzymes.

Publications

On the role of a conserved, potentially helix-breaking residue in the tRNA-binding alpha-helix of archaeal CCA-adding enzymes.

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