Association Between Archaeal Prolyl- and Leucyl-tRNA Synthetases Enhances tRNA\u3csup\u3ePro\u3c/sup\u3e Aminoacylation

Aminoacyl-tRNA synthetase-containing complexes have been identified in different eukaryotes, and their existence has also been suggested in some Archaea. To investigate interactions involving aminoacyl-tRNA synthetases in Archaea, we undertook a yeast two-hybrid screen for interactions between Methanothermobacter thermautotrophicus proteins using prolyl-tRNA synthetase (ProRS) as the bait. Interacting proteins identified included components of methanogenesis, protein-modifying factors, and leucyl-tRNA synthetase (LeuRS). The association of ProRS with LeuRS was confirmed in vitro by native gel electrophoresis and size exclusion chromatography. Determination of the steady-state kinetics of tRNAPro charging showed that the catalytic efficiency (kcat/Km) of ProRS increased 5-fold in the complex with LeuRS compared with the free enzyme, whereas the Km for proline was unchanged. No significant changes in the steady-state kinetics of LeuRS aminoacylation were observed upon the addition of ProRS. These findings indicate that ProRS and LeuRS associate in M. thermautotrophicus and suggest that this interaction contributes to translational fidelity by enhancing tRNA aminoacylation by ProRS

Functional Association Between Three Archaeal Aminoacyl-tRNA Synthetases

Aminoacyl-tRNA synthetases (aaRSs) are responsible for attaching amino acids to their cognate tRNAs during protein synthesis. In eukaryotes aaRSs are commonly found in multi-enzyme complexes, although the role of these complexes is still not completely clear. Associations between aaRSs have also been reported in archaea, including a complex between prolyl-(ProRS) and leucyl-tRNA synthetases (LeuRS) in Methanothermobacter thermautotrophicus that enhances tRNAPro aminoacylation. Yeast two-hybrid screens suggested that lysyl-tRNA synthetase (LysRS) also associates with LeuRS in M. thermautotrophicus. Co-purification experiments confirmed that LeuRS, LysRS, and ProRS associate in cell-free extracts. LeuRS bound LysRS and ProRS with a comparable KD of about 0.3–0.9 μm, further supporting the formation of a stable multi-synthetase complex. The steady-state kinetics of aminoacylation by LysRS indicated that LeuRS specifically reduced the Km for tRNALys over 3-fold, with no additional change seen upon the addition of ProRS. No significant changes in aminoacylation by LeuRS or ProRS were observed upon the addition of LysRS. These findings, together with earlier data, indicate the existence of a functional complex of three aminoacyl-tRNA synthetases in archaea in which LeuRS improves the catalytic efficiency of tRNA aminoacylation by both LysRS and ProRS

Aspartyl-tRNA Synthetase is the Target of Peptidenucleotide Antibiotic Microcin C

Microcin C is a ribosome-synthesized heptapeptide that contains a modified adenosine monophosphate covalently attached to the C-terminal aspartate. Microcin C is a potent inhibitor of bacterial cell growth. Based on the in vivo kinetics of inhibition of macromolecular synthesis, Microcin C targets translation, through a mechanism that remained undefined. Here, we show that Microcin C is a subject of specific degradation inside the sensitive cell. The product of degradation, a modified aspartyl-adenylate containing an N-acylphosphoramidate linkage, strongly inhibits translation by blocking the function of aspartyl-tRNA synthetase

tRNAs: Cellular Barcodes for Amino Acids

The role of tRNA in translating the genetic code has received considerable attention over the last 50 years, and we now know in great detail how particular amino acids are specifically selected and brought to the ribosome in response to the corresponding mRNA codon. Over the same period, it has also become increasingly clear that the ribosome is not the only destination to which tRNAs deliver amino acids, with processes ranging from lipid modification to antibiotic biosynthesis all using aminoacyl‐tRNAs as substrates. Here we review examples of alternative functions for tRNA beyond translation, which together suggest that the role of tRNA is to deliver amino acids for a variety of processes that includes, but is not limited to, protein synthesis

Aminoacyl-tRNA Synthesis in Archaea: Different but Not Unique

Accurate aminoacyl‐tRNA synthesis is essential for correct translation of the genetic code in all organisms. Whereas many aspects of this process are conserved, others display a surprisingly high level of divergence from the canonical Escherichia coli model system. These differences are most pronounced in archaea where novel mechanisms have recently been described for aminoacylating tRNAs with asparagine, cysteine, glutamine and lysine. Whereas these mechanisms were initially assumed to be uniquely archaeal, both the alternative asparagine and lysine pathways have subsequently been demonstrated in numerous bacteria. Similarly, studies of the means by which archaea insert the rare amino acid selenocysteine in response to UGA stop codons have helped provide a better understanding of both archaeal and eukaryal selenoprotein synthesis. Most recently a new co‐translationally inserted amino acid, pyrrolysine, has been found in archaea although again there is some suggestion that it may also be present in bacteria. Thus, whereas archaea contain a preponderance of non‐canonical aminoacyl‐tRNA synthesis systems most are also found elsewhere albeit less frequently

Transfer RNA Identity Contributes to Transition State Stabilization During Aminoacyl-tRNA Synthesis

Sequence-specific interactions between aminoacyl-tRNA synthetases and their cognate tRNAs ensure both accurate RNA recognition and the efficient catalysis of aminoacylation. The effects of tRNATrP variants on the aminoacylation reaction catalyzed by wild-type Escherichia coli tryptophanyl-tRNA synthe-tase (TrpRS) have now been investigated by stopped-flow fluorimetry, which allowed a pre-steady-state analysis to be undertaken. This showed that tRNATrP identity has some effect on the ability of tRNA to bind the reaction intermediate TrpRS-tryptophanyl-adenylate, but predominantly affects the rate at which trypto-phan is transferred from TrpRS-tryptophanyl ade-nylate to tRNA. Use of the binding (KtRNA) and rate constants (k4) to determine the energetic levels of the various species in the aminoacylation reaction showed a difference of ∼2 kcal mol-1 in the barrier to transition state formation compared to wild-type for both tRNATrP A→C73 an

Discrimination of Cognate and Non-Cognate Substrates at the Active Site of Class I Lysyl-tRNA Synthetase

The aminoacyl-tRNA synthetases are divided into two unrelated structural classes, with lysyl-tRNA synthetase (LysRS) being the only enzyme represented in both classes. On the basis of the structure of l-lysine complexed with Pyrococcus horikoshii class I LysRS (LysRS1) and homology to glutamyl-tRNA synthetase (GluRS), residues implicated in amino acid recognition and noncognate substrate discrimination were systematically replaced in Borrelia burgdorferi LysRS1. The catalytic efficiency of steady-state aminoacylation (kcat/KM) with lysine by LysRS1 variants fell by 1−4 orders of magnitude compared to that of the wild type. Disruption of putative hydrogen bonding interactions through replacement of G29, T31, and Y269 caused up to 1500-fold reductions in kcat/KM, similar to changes previously observed for comparable variants of class II LysRS (LysRS2). Replacements of W220 and H242, both of which are implicated in hydrophobic interactions with the side chain of lysine, resulted in more dramatic changes with up to 40000-fold reductions in kcat/KM observed. This indicates that the more compact LysRS1 active site employs both electrostatic and hydrophobic interactions during lysine discrimination, explaining the ability of LysRS1 to discriminate against noncognate substrates accepted by LysRS2. Several of the LysRS1 variants were found to be more specific than the wild type with respect to noncognate amino acid recognition but less efficient in cognate aminoacylation. This indicates that LysRS1 compromises between efficient catalysis and substrate discrimination, in contrast to LysRS2 which is considerably more effective in catalysis but is less specific than its class I counterpart

tRNAs: cellular barcodes for amino acids

AbstractThe role of tRNA in translating the genetic code has received considerable attention over the last 50 years, and we now know in great detail how particular amino acids are specifically selected and brought to the ribosome in response to the corresponding mRNA codon. Over the same period, it has also become increasingly clear that the ribosome is not the only destination to which tRNAs deliver amino acids, with processes ranging from lipid modification to antibiotic biosynthesis all using aminoacyl-tRNAs as substrates. Here we review examples of alternative functions for tRNA beyond translation, which together suggest that the role of tRNA is to deliver amino acids for a variety of processes that includes, but is not limited to, protein synthesis