CRYSTAL STRUCTURE DETERMINATION OF THE ALANYL-TRNA SYNTHETASE AND ITS COMPLEXES

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Aminoacyl-tRNA synthetases (aaRSs) are a family of 20 essential enzymes responsible for attaching the 20 amino acids to their cognate transfer-RNAs (tRNAs) in a highly specific reaction, thereby affecting the translation of the genetic code in all living cells. Alanyl-tRNA snythetase (AlaRS) attaches alanine onto tRNAAla in two-steps. First, enzyme-bound alanine is activated by ATP, then the alanyl-adenylate moiety is transferred to enzyme-bound tRNAAla, releasing AMP and pyrophosphate. Among aaRSs, AlaRS is functionally unique. For example, AlaRS requires no more than a few (seven) base pairs in the tRNA acceptor stem for specific aminoacylation with alanine and is indifferent toward the tRNA's anticodon sequence. The major determinant of the identity of tRNAAla is a single wobble base pair, G3:U70, located in the acceptor helix. Mutations in this base pair prevent aminoacylation in vitro and in vivo and its inclusion in non-cognate tRNAs enables them to accept alanine. We want to reveal the structural basis of these unique functional properties of AlaRS. Of the 20 aaRSs, AlaRS remains to be the only synthetase without a known crystal structure. This is largely due to difficulty in crystallizing the enzyme. Using a novel high-throughput crystallization approach, we recently obtained native and sel-Met-labelled crystals of a catalytic fragment of AlaRS from the extreme thermophile Aquifex aeolicus. The crystals diffract to 3.0 ¿¿on in-house equipment and fall in the space group P21212, with one molecule in the asymmetric unit. Complex crystals with RNA are prepared by diffusing small RNA substrates into exiting crystals of the free enzyme. Our goal is to determine the de novo crystal structures of the AlaRS catalytic fragment and complexes with RNA using MAD methods. To that end, we hope to collect the necessary Se-MAD data at SSRL.