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Evolution of acceptor stem tRNA recognition by class II prolyl-tRNA synthetase.

An S, Barany G, Musier-Forsyth K - Nucleic Acids Res. (2008)

Bottom Line: Incorporation of site-specific 2'-deoxynucleotides, as well as phosphorothioate and methylphosphonate modifications within the tRNA acceptor stem revealed an extensive network of interactions with specific functional groups proximal to the first base pair and the discriminator base.Therefore, in contrast to the bacterial system, backbone-specific interactions contribute significantly more to tRNA recognition by the human enzyme than base-specific interactions.Taken together with previous studies, these data show that ProRS-tRNA acceptor stem interactions have co-adapted through evolution from a mechanism involving 'direct readout' of nucleotide bases to one relying primarily on backbone-specific 'indirect readout'.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of Minnesota, Minneapolis, MN 55455, USA.

ABSTRACT
Aminoacyl-tRNA synthetases (AARS) are an essential family of enzymes that catalyze the attachment of amino acids to specific tRNAs during translation. Previously, we showed that base-specific recognition of the tRNA(Pro) acceptor stem is critical for recognition by Escherichia coli prolyl-tRNA synthetase (ProRS), but not for human ProRS. To further delineate species-specific differences in acceptor stem recognition, atomic group mutagenesis was used to probe the role of sugar-phosphate backbone interactions in recognition of human tRNA(Pro). Incorporation of site-specific 2'-deoxynucleotides, as well as phosphorothioate and methylphosphonate modifications within the tRNA acceptor stem revealed an extensive network of interactions with specific functional groups proximal to the first base pair and the discriminator base. Backbone functional groups located at the base of the acceptor stem, especially the 2'-hydroxyl of A66, are also critical for aminoacylation catalytic efficiency by human ProRS. Therefore, in contrast to the bacterial system, backbone-specific interactions contribute significantly more to tRNA recognition by the human enzyme than base-specific interactions. Taken together with previous studies, these data show that ProRS-tRNA acceptor stem interactions have co-adapted through evolution from a mechanism involving 'direct readout' of nucleotide bases to one relying primarily on backbone-specific 'indirect readout'.

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Sequence of the semi-synthetic A57G human tRNAPro variant used in these studies. The tRNA was constructed by annealing an in vitro transcribed 5′-57-mer with a chemically synthesized 3′-16-mer containing site-specific backbone modifications.
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Figure 1: Sequence of the semi-synthetic A57G human tRNAPro variant used in these studies. The tRNA was constructed by annealing an in vitro transcribed 5′-57-mer with a chemically synthesized 3′-16-mer containing site-specific backbone modifications.

Mentions: We previously demonstrated that E. coli ProRS can efficiently aminoacylate semi-synthetic E. coli tRNAs prepared by annealing two RNA fragments (19, 29). In the present work, semi-synthetic human tRNAPro was constructed by annealing an in vitro transcribed 5′-57-mer with a chemically synthesized 3′-16-mer (Figure 1). The wild-type semi-synthetic construct is a good substrate for human ProRS, despite the fact that it contains a break in the phosphodiester backbone in the TΨC loop. The efficiency of aminoacylation of semi-synthetic human tRNAPro was similar to that of in vitro transcribed full-length human tRNAPro and aminoacylation assays were performed as previously described (29). To investigate the contribution of specific acceptor stem backbone functional groups in aminoacylation by human ProRS, three different modifications were incorporated into semi-synthetic tRNAs using automated chemical synthesis: 2′-deoxy, phosphorothioate and methylphosphonate. Substitutions were incorporated into positions 63–75 of the 3′-strand of the tRNA, which can be arbitrarily divided into three local regions: a single-strand top region, positions 73–76; a double-stranded acceptor stem region, positions 66–72; and a double-stranded TΨC stem region, position 63–65.Figure 1.


Evolution of acceptor stem tRNA recognition by class II prolyl-tRNA synthetase.

An S, Barany G, Musier-Forsyth K - Nucleic Acids Res. (2008)

Sequence of the semi-synthetic A57G human tRNAPro variant used in these studies. The tRNA was constructed by annealing an in vitro transcribed 5′-57-mer with a chemically synthesized 3′-16-mer containing site-specific backbone modifications.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2377447&req=5

Figure 1: Sequence of the semi-synthetic A57G human tRNAPro variant used in these studies. The tRNA was constructed by annealing an in vitro transcribed 5′-57-mer with a chemically synthesized 3′-16-mer containing site-specific backbone modifications.
Mentions: We previously demonstrated that E. coli ProRS can efficiently aminoacylate semi-synthetic E. coli tRNAs prepared by annealing two RNA fragments (19, 29). In the present work, semi-synthetic human tRNAPro was constructed by annealing an in vitro transcribed 5′-57-mer with a chemically synthesized 3′-16-mer (Figure 1). The wild-type semi-synthetic construct is a good substrate for human ProRS, despite the fact that it contains a break in the phosphodiester backbone in the TΨC loop. The efficiency of aminoacylation of semi-synthetic human tRNAPro was similar to that of in vitro transcribed full-length human tRNAPro and aminoacylation assays were performed as previously described (29). To investigate the contribution of specific acceptor stem backbone functional groups in aminoacylation by human ProRS, three different modifications were incorporated into semi-synthetic tRNAs using automated chemical synthesis: 2′-deoxy, phosphorothioate and methylphosphonate. Substitutions were incorporated into positions 63–75 of the 3′-strand of the tRNA, which can be arbitrarily divided into three local regions: a single-strand top region, positions 73–76; a double-stranded acceptor stem region, positions 66–72; and a double-stranded TΨC stem region, position 63–65.Figure 1.

Bottom Line: Incorporation of site-specific 2'-deoxynucleotides, as well as phosphorothioate and methylphosphonate modifications within the tRNA acceptor stem revealed an extensive network of interactions with specific functional groups proximal to the first base pair and the discriminator base.Therefore, in contrast to the bacterial system, backbone-specific interactions contribute significantly more to tRNA recognition by the human enzyme than base-specific interactions.Taken together with previous studies, these data show that ProRS-tRNA acceptor stem interactions have co-adapted through evolution from a mechanism involving 'direct readout' of nucleotide bases to one relying primarily on backbone-specific 'indirect readout'.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of Minnesota, Minneapolis, MN 55455, USA.

ABSTRACT
Aminoacyl-tRNA synthetases (AARS) are an essential family of enzymes that catalyze the attachment of amino acids to specific tRNAs during translation. Previously, we showed that base-specific recognition of the tRNA(Pro) acceptor stem is critical for recognition by Escherichia coli prolyl-tRNA synthetase (ProRS), but not for human ProRS. To further delineate species-specific differences in acceptor stem recognition, atomic group mutagenesis was used to probe the role of sugar-phosphate backbone interactions in recognition of human tRNA(Pro). Incorporation of site-specific 2'-deoxynucleotides, as well as phosphorothioate and methylphosphonate modifications within the tRNA acceptor stem revealed an extensive network of interactions with specific functional groups proximal to the first base pair and the discriminator base. Backbone functional groups located at the base of the acceptor stem, especially the 2'-hydroxyl of A66, are also critical for aminoacylation catalytic efficiency by human ProRS. Therefore, in contrast to the bacterial system, backbone-specific interactions contribute significantly more to tRNA recognition by the human enzyme than base-specific interactions. Taken together with previous studies, these data show that ProRS-tRNA acceptor stem interactions have co-adapted through evolution from a mechanism involving 'direct readout' of nucleotide bases to one relying primarily on backbone-specific 'indirect readout'.

Show MeSH
Related in: MedlinePlus