Dissection of a class II tRNA synthetase: Determinants for minihelix recognition are tightly associated with domain for amino acid activation

Abstract

The ten class II aminoacyl-tRNA synthetases are large homo- and hetero-oligomeric proteins that share three conserved sequence motifs. Within this class, Escherichia coli alanyl-tRNA synthetase is the only homotetramer and is comprised of subunits of 875 amino acids. The enzyme aminoacylates sequence-specific RNA oligonucleotides that recreate as few as four base pairs of the acceptor stem of tRNA(Ala). A monomeric 461 amino acid N-terminal fragment (461N) was previously shown to have full adenylate synthesis activity. However, fragment 461N has significant, but reduced, efficiency of charging of tRNA(Ala), when compared to native enzyme [Ho, C., Jasin, M., & Schimmel, P. (1985) Science 229, 389-393]. We show here that, in contrast, the fragment and the native enzyme aminoacylate a 12 base pair acceptor-T psi C stem minihelix and a four base pair RNA tetraloop with the same efficiency. We also show that fragment 461N makes footprint contacts both on and outside the acceptor helix of bound tRNA(Ala). With one possible exception, the contacts observed with fragment 461N are identical to those seen with the native enzyme. In spite of contacts outside the acceptor helix, fragment 461N charges the native tRNA, minihelix, and tetraloop with similar efficiency. Thus, all minihelix contacts required for activation for charging are tightly associated with the adenylate synthesis domain and, at least in the fragment, are not influenced by additional RNA-protein contacts outside the minihelix domain.(ABSTRACT TRUNCATED AT 250 WORDS)

RNA recognition by designed peptide fusion creates "artificial" tRNA synthetase

“…1B). Although the native enzyme is tetrameric, fragment 368N is a monomer that catalyzes synthesis of alanyl adenylate with the same efficiency as that of the native enzyme (34,40). Fragment 368N has no activity for aminoacylation or tRNA binding.…”

“…As a consequence, 461N aminoacylates microhelix Ala with the same catalytic efficiency as does the native enzyme. [The native enzyme has a smaller K m for charging tRNA Ala than does 461N because of contacts with tRNA Ala that lie outside the acceptor arm that are contributed by portions of AlaRS that lie on the C-terminal side of D461 and therefore are missing from 461N (40).] Thus, in our work we sought to replace the 93-aa segment from T369 to D461, which is needed for acceptor helix contacts.…”

Structure-specific tRNA Determinants for Editing a Mischarged Amino Acid

“…Aminoacylation and Deacylation Assays-Aminoacylation assays were performed at 37°C as described previously (29) in charging buffer (50 mM HEPES, pH 7.5, 20 mM KCl, 0.1 mg/ml bovine serum albumin, 20 mM ␤-mercaptoethanol, and10 mM MgCl 2 ). Mischarging with wildtype AlaRS (5 M) was done in the presence of tRNA Ala transcript (5 M) or minihelix Ala (15 M) and [ 3 H]Gly (10.7 M) in charging buffer at 37°C.…”

Substrate Recognition by Novel Family of Amino Acid:[Carrier Protein] Ligases

“…In that case, low level of residual tRNA acylation activity would be expected, and possibly tRNA aminoacylation activity could be restored by providing tRNA-binding domain in cis. It was demonstrated for AlaRS, 35 MetRS 36 and SerRS 37 that their catalytic domain devoid of tRNA-binding domain retains residual tRNA aminoacylation activity. On the other hand, aaRS are modular enzymes and it is generally accepted that modern aaRSs originated from primitive catalytic cores capable of amino acid activation by subsequent acquisition of additional domains, such as tRNA-binding and editing domains.…”