Design of an Active Fragment of a Class II Aminoacyl-tRNA Synthetase and Its Significance for Synthetase Evolution

Abstract

Primordial aminoacyl-tRNA synthetases (aaRSs) based on the Rossman nucleotide binding fold of class I enzymes or the seven-stranded antiparallel beta-sheet fold of class II enzymes have been proposed to predate the contemporary aaRS. As part of an inquiry into class II aaRS evolution, the individual domains of the homodimeric Escherichia coli histidyl-tRNA synthetase (HisRS) were separately expressed and purified to determine their individual contributions to catalysis. A 320-residue fragment (Ncat HisRS) truncated immediately following motif 3 catalyzes both the specific aminoacylation of tRNA and pyrophosphate exchange, albeit less efficiently than the full-length enzyme. Ncat HisRS showed no mischarging of noncognate tRNAs but exhibited reduced selectivity for the C73 discriminator base, a principal aminoacylation determinant for histidine tRNAs. Size exclusion chromatography showed that Ncat HisRS is monomeric, indicating that the C-terminal domain is essential for maintaining the dimeric structure of the enzyme. The stably folded C-terminal domain (Cter HisRS) was inactive for both reactions and did not enhance the activity of Ncat HisRS when added in trans. The fusion of one or more accessory domains to a primordial catalytic domain may therefore have been a critical evolutionary step by which aminoacyl-tRNA synthetases acquired increased catalytic efficiency and substrate specificity.

Tryptophanyl-tRNA Synthetase Urzyme

“…The active fraction is obtained from the fitted parameters (24) 32 PP i Exchange Assays for Tryptophan Activation-Samples were assayed using the traditional 32 P-pyrophosphate exchange as described previously (25,26) except that a 3-fold reduction of background 32 P counts was achieved by collecting the charcoal containing labeled ATP on disposable spin columns, washing, and eluting the bound ATP with 50 l of pyridine at 37°C (19). ATP and tryptophan were first depleted from the assay buffer to determine ATP-and tryptophan-dependent Michaelis-Menten constants.…”

“…The domain structures of aminoacyl-tRNA synthetases motivated previous experimental truncations to isolate active catalytic fragments (32)(33)(34)(35). These efforts focused mainly on cleaving between the catalytic and anticodon-binding domains, a widely documented approach applied routinely only to proteins whose domain structures can be readily identified (36 -38).…”

“…These efforts focused mainly on cleaving between the catalytic and anticodon-binding domains, a widely documented approach applied routinely only to proteins whose domain structures can be readily identified (36 -38). For example, an N-terminal 320-residue HisRS fragment (ϳ60% of the native monomer) that retains an intact catalytic domain is 10 3 -fold less active without its deleted anticodon-binding domain (32). Such truncations are far less aggressive than those made to cognate tRNAs, which ultimately resulted in identifying minihelices ϳ30% the size of intact tRNAs (39,40) that could be charged by several aaRS.…”

“…Several assays on inactive cleaved preparations showed no activity, ruling out activity arising from Factor Xa. The digestion mixture was used directly for 32 PP i exchange assays, showing that digestion also released a cryptic tryptophan activating activity from the fusion protein.…”

Aminoacylating Urzymes Challenge the RNA World Hypothesis

“…Moreover, Koonin estimates that assembling the replicative fidelity necessary for an RNA-only origin would require multiple universes (3). tRNA aminoacylation, the defining reaction in codon-dependent translation, has not previously been demonstrated for aaRS 2 fragments smaller than intact catalytic domains (4,5). Experimental acylation of mini-and microhelices derived from tRNAs (6, 7) has been shown, however, for several intact aaRSs, suggesting that aaRS catalytic domains might have acylated cognate tRNAs using an earlier "operational RNA code" (8) focused on bases in the 3Ј acceptor stems.…”