title: Assembly of a catalytic unit for RNA microhelix aminoacylation using nonspecific RNA binding domains.
abstract: An assembly of a catalytic unit for aminoacylation of an RNA microhelix is demonstrated here. This assembly may recapitulate a step in the historical development of tRNA synthetases. The class-defining domain of a tRNA synthetase is closely related to the primordial enzyme that catalyzed synthesis of aminoacyl adenylate. RNA binding elements are imagined to have been added so that early RNA substrates could be docked proximal to the activated amino acid. RNA microhelices that recapitulate the acceptor stem of modern tRNAs are potential examples of early substrates. In this work, we examined a fragment of Escherichia coli alanyl-tRNA synthetase, which catalyzes aminoacyl adenylate formation but is virtually inactive for catalysis of RNA microhelix aminoacylation. Fusion to the fragment of either of two unrelated nonspecific RNA binding domains activated microhelix aminoacylation. Although the fusion proteins lacked the RNA sequence specificity of the natural enzyme, their activity was within 1-2 kcal.mol(-1) of a truncated alanyl-tRNA synthetase that has aminoacylation activity sufficient to sustain cell growth. These results show that, starting with an activity for adenylate synthesis, barriers are relatively low for building catalytic units for aminoacylation of RNA helices.
authors.affiliation: Skaggs Institute for Chemical Biology, Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.
authors.forename: J W
authors.initials: JW
authors.lastname: Chihade
authors.forename: P
authors.initials: P
authors.lastname: Schimmel
mesh_terms: D000215:Acylation; D000604:Amino Acyl-tRNA Synthetases / Q000378:metabolism*; D001483:Base Sequence; D020134:Catalytic Domain; D017931:DNA Primers; D007700:Kinetics; D009690:Nucleic Acid Conformation; D012313:RNA / Q000737:chemistry / Q000378:metabolism*
chemical_list: D017931:DNA Primers; D012313:RNA; D000604:Amino Acyl-tRNA Synthetases