Cysteinyl-tRNA formation and prolyl-tRNA synthetase

AbstractAminoacyl-tRNA (AA-tRNA) formation is a key step in protein biosynthesis. This reaction is catalyzed with remarkable accuracy by the AA-tRNA synthetases, a family of 20 evolutionarily conserved enzymes. The lack of cysteinyl-tRNA (Cys-tRNA) synthetase in some archaea gave rise to the discovery of the archaeal prolyl-tRNA (Pro-tRNA) synthetase, an enzyme capable of synthesizing Pro-tRNA and Cys-tRNA. Here we review our current knowledge of this fascinating process

Computational Insights into the High-Fidelily Catalysis of Aminoacyl-tRNA Synthetases

Obtaining insights into the catalytic function of enzymes is an important area of research due to their widespread applications in the biotechnology and pharmaceutical industries. Among these enzymes, the aminoacyl-tRNA synthetases (aaRSs) are known for their remarkable fidelity in catalyzing the aminoacylation reactions of tRNA in protein biosynthesis. Despite the exceptional execution of this critical function, mechanistic details of the reactions catalyzed by aminoacyl-tRNA synthetases remain elusive demonstrating the obvious need to explore their remarkable chemistry. During the PhD studies reported in this thesis the mechanism of aminoacylation, pre-transfer editing and post-transfer editing catalyzed by different aaRS have been established using multi-scale computational enzymology. In the first two chapters a detailed information about aaRS and the addressed questions was given in addition to an overview of the used computational methodology currently used to investigate the enzymatic mechanisms. The aminoacylation mechanism of threonine by Threonyl-tRNA synthetases, glutamine by Glutaminyl-tRNA synthetases and glutamate by Glutamyl-tRNA synthetases have been clearly unveiled in chapter 3 and 4. Also, valuable information regarding the role of cofactors and active site residues has been obtained. While investigating the post-transfer editing mechanisms, which proceed in a remote and distinct active site, two different scenarios were experimentally suggested for two types of threonyltRNA synthetase species to correct the misacylation of the structurally related serine. We explored these two mechanisms as in chapters 5 and 6. Moreover, the synthetic site in which the aminoacylation reaction is catalyzed, is also responsible for a second type of proofreading reaction called pre-transfer editing mechanism. In chapter 7, this latter mechanism has been elucidated for both Seryl-tRNA synthetases and Isoleucyl-tRNA synthetases against their non-cognate substrates cysteine and valine, respectively. In chapter 8, an assessment QM/MM study using a variety of DFT functionals to represent the chemically active layer in aminoacylation mechanism of the unnatural amino acid ß-Hydroxynorvaline as catalyzed by Threonyl-tRNA synthetase has been carried out. Overall, it was found that substrateassisted mechanisms are a common pathway for these enzymes. One important application of such information is to establish the criteria required for any candidate to inhibit the catalytic functions of aaRS, which was applied in chapter 9 to screen potential competitive inhibitors able to efficiently block the bacterial Threonyl-tRNA synthetases. The investigations reported herein should provide atomistic details into the fundamental catalytic mechanisms of the ubiquitous and ancient aaRS enzymes. Consequently, they will also help enable a much-needed deeper understanding of the underlying chemical principles of catalysis in general

Stereochemical errors and their implications for molecular dynamics simulations

<p>Abstract</p> <p>Background</p> <p>Biological molecules are often asymmetric with respect to stereochemistry, and correct stereochemistry is essential to their function. Molecular dynamics simulations of biomolecules have increasingly become an integral part of biophysical research. However, stereochemical errors in biomolecular structures can have a dramatic impact on the results of simulations.</p> <p>Results</p> <p>Here we illustrate the effects that chirality and peptide bond configuration flips may have on the secondary structure of proteins throughout a simulation. We also analyze the most common sources of stereochemical errors in biomolecular structures and present software tools to identify, correct, and prevent stereochemical errors in molecular dynamics simulations of biomolecules.</p> <p>Conclusions</p> <p>Use of the tools presented here should become a standard step in the preparation of biomolecular simulations and in the generation of predicted structural models for proteins and nucleic acids.</p

Characterizing the Editing Modes of Duplicated Leucyl-Trna Synthetases in the Archaea Sulfolobus Islandicus

Aminoacyl-transfer RNA synthetases (aaRSs) catalyze the attachment of an amino acid to the 3’-end of a cognate tRNA in a two-step reaction, fulfilling a pivotal role in RNA translation by providing “charged” tRNA for protein synthesis. Synthetases possess a catalytic core where amino acids are activated via adenylation with the subsequent transfer of that activated amino acid to the acceptor adenosine at position 76 of the tRNA sequence.1,2 The fidelity of tRNA aminoacylation is essential for faithful protein synthesis and requires the aaRS to discriminate against chemically and structurally similar amino acids, though some studies suggest that aaRS-dependent errors can be beneficial to some microbial species.3The meticulous accuracy of aaRS is dependent in part upon amino acid editing mechanisms. The aliphatic synthetases rely on hydrolytic editing activity to clear mischarged tRNA (post-transfer), performed in the connective polypeptide 1 (CP1) domain, distinct from the synthetic aminoacylation site. A second editing pathway hydrolyzes the aminoacyl adenylate intermediate at the synthetic site before transfer of the amino acid to the tRNA (pre-transfer).4 Both pre- and post-transfer editing capacity can co-exist in a single aaRS, though one is generally dominant in standard conditions. However, when an editing pathway is unavailable, the secondary pathway may be stimulated to suppress aaRS infidelity.5Bioinformatic analyses identified two distinct leucyl-tRNA synthetase genes within all genomes of the Archaeal family Sulfolobaceae.6 One copy, named LeuRS-I, has substitutions of key amino acids within its CP1 editing domain that would be expected to eliminate its ability to post-transfer edit mischarged tRNALeu, which could bring about variation within the proteome of these extremophiles. The other copy, LeuRS-F, contains canonical active sites in both the catalytic core and CP1 domain.6 Biochemical analysis of the paralogs within Sulfolobus islandicus supports the hypothesis that LeuRS-F, and not LeuRS-I, functions as an essential tRNA synthetase that faithfully charges leucine to tRNALeu. Remarkably, the ubiquitous conservation of LeuRS-I across the Sulfolobaceae, despite its inability to perform canonical synthetase activity, indicates an alternative, non-canonical function of this paralog. Further analysis of the charging and hydrolytic editing capability of these paralogs was performed by selective mutation of key residues in the CP1 domain of LeuRS-F to match those in LeuRS-I. Interestingly, elimination of the post-transfer editing capability of LeuRS-F does not result in mischarged tRNA, suggesting stimulation of a pre-transfer editing mechanism not commonly observed in LeuRS. Additional enzymatic analysis shows activation of non-cognate amino acids by both LeuRSs, a secondary tRNA dependent activity to prevent mischarging, and a defunct post-transfer editing capability for the LeuRS-F CP1-domain mutant

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

The physiological target for LeuRS translational quality control is norvaline

The fidelity of protein synthesis depends on the capacity of aminoacyl-tRNA synthetases (AARSs) to couple only cognate amino acid-tRNA pairs. If amino acid selectivity is compromised, fidelity can be ensured by an inherent AARS editing activity that hydrolyses mischarged tRNAs. Here we show that the editing activity of Escherichia coli leucyl-tRNA synthetase (EcLeuRS) is not required to prevent incorrect isoleucine incorporation. Rather, as shown by kinetic, structural and in vivo approaches, the prime biological function of LeuRS editing is to prevent mis-incorporation of the non-standard amino acid norvaline. This conclusion follows from a reassessment of the discriminatory power of LeuRS against isoleucine and the demonstration that a LeuRS editing- deficient E. coli strain grows normally in high concentrations of isoleucine but not under oxygen deprivation conditions when norvaline accumulates to substantial levels. Thus, AARS- based translational quality control is a key feature for bacterial adaptive response to oxygen deprivation. The non-essential role for editing under normal bacterial growth has important implications for the development of resistance to antimicrobial agents targeting the LeuRS editing site

Modular pathways for editing non-cognate amino acids by human cytoplasmic leucyl-tRNA synthetase

To prevent potential errors in protein synthesis, some aminoacyl-transfer RNA (tRNA) synthetases have evolved editing mechanisms to hydrolyze misactivated amino acids (pre-transfer editing) or misacylated tRNAs (post-transfer editing). Class Ia leucyl-tRNA synthetase (LeuRS) may misactivate various natural and non-protein amino acids and then mischarge tRNALeu. It is known that the fidelity of prokaryotic LeuRS depends on multiple editing pathways to clear the incorrect intermediates and products in the every step of aminoacylation reaction. Here, we obtained human cytoplasmic LeuRS (hcLeuRS) and tRNALeu (hctRNALeu) with high activity from Escherichia coli overproducing strains to study the synthetic and editing properties of the enzyme. We revealed that hcLeuRS could adjust its editing strategy against different non-cognate amino acids. HcLeuRS edits norvaline predominantly by post-transfer editing; however, it uses mainly pre-transfer editing to edit α-amino butyrate, although both amino acids can be charged to tRNALeu. Post-transfer editing as a final checkpoint of the reaction was very important to prevent mis-incorporation in vitro. These results provide insight into the modular editing pathways created to prevent genetic code ambiguity by evolution

Translational Fidelity, Mistranslation, and the Cellular Responses to Stress

Faithful translation of mRNA into the corresponding polypeptide is a complex multistep process, requiring accurate amino acid selection, transfer RNA (tRNA) charging and mRNA decoding on the ribosome. Key players in this process are aminoacyl-tRNA synthetases (aaRSs), which not only catalyse the attachment of cognate amino acids to their respective tRNAs, but also selectively hydrolyse incorrectly activated non-cognate amino acids and/or misaminoacylated tRNAs. This aaRS proofreading provides quality control checkpoints that exclude non-cognate amino acids during translation, and in so doing helps to prevent the formation of an aberrant proteome. However, despite the intrinsic need for high accuracy during translation, and the widespread evolutionary conservation of aaRS proofreading pathways, requirements for translation quality control vary depending on cellular physiology and changes in growth conditions, and translation errors are not always detrimental. Recent work has demonstrated that mistranslation can also be beneficial to cells, and some organisms have selected for a higher degree of mistranslation than others. The aims of this Review Article are to summarize the known mechanisms of protein translational fidelity and explore the diversity and impact of mistranslation events as a potentially beneficial response to environmental and cellular stress

Naturally Occurring Isoleucyl-tRNA Synthetase without tRNA-dependent Pre-transfer Editing

Isoleucyl-tRNA synthetase (IleRS) is unusual among aminoacyl-tRNA synthetases in having a tRNA-dependent pre-transfer editing activity. Alongside the typical bacterial IleRS (such as Escherichia coli IleRS), some bacteria also have the enzymes (eukaryote-like) that cluster with eukaryotic IleRSs and exhibit low sensitivity to the antibiotic mupirocin. Our phylogenetic analysis suggests that the ileS1 and ileS2 genes of contemporary bacteria are the descendants of genes that might have arisen by an ancient duplication event before the separation of bacteria and archaea. We present the analysis of evolutionary constraints of the synthetic and editing reactions in eukaryotic/eukaryote-like IleRSs, which share a common origin but diverged through adaptation to different cell environments. The enzyme from the yeast cytosol exhibits tRNA-dependent pre-transfer editing analogous to E. coli IleRS. This argues for the presence of this proofreading in the common ancestor of both IleRS types and an ancient origin of the synthetic site-based quality control step. Yet surprisingly, the eukaryote-like enzyme from Streptomyces griseus IleRS lacks this capacity; at the same time, its synthetic site displays the 10(3)-fold drop in sensitivity to antibiotic mupirocin relative to the yeast enzyme. The discovery that pre-transfer editing is optional in IleRSs lends support to the notion that the conserved post-transfer editing domain is the main checkpoint in these enzymes. We substantiated this by showing that under error-prone conditions S. griseus IleRS is able to rescue the growth of an E. coli lacking functional IleRS, providing the first evidence that tRNA-dependent pre-transfer editing in IleRS is not essential for cell viability