Reversible inactivation of yeast mitochondrial phenylalanyl-tRNA synthetase under oxidative stress

Background

Under oxidative stress cytoplasmic aminoacyl-tRNA synthetase (aaRSs) substrate specificity can be compromised, leading to tRNA mischarging and mistranslation of the proteome. Whether similar processes occur in mitochondria, which are major cellular sources of reactive oxygen species (ROS), is unknown. However, relaxed substrate specificity in yeast mitochondrial phenylalanyl-tRNA synthetase (ScmitPheRS) has been reported to increase tRNA mischarging and blocks mitochondrial biogenesis.

Classical molecular dynamics simulation of seryl tRNA synthetase and threonyl tRNA synthetase bound with tRNA and aminoacyl adenylate

Lacunae of understanding exist concerning the active site organization during the charging step of the aminoacylation reaction. We present here a molecular dynamics simulation study of the dynamics of the active site organization during charging step of subclass IIa dimeric SerRS from Thermus thermophilus (^ttSerRS) bound with ^tttRNA^Ser and dimeric ThrRS from Escherichia coli (^ecThrRS) bound with ^ectRNA^Thr. The interactions between the catalytically important loops and tRNA contribute to the change in dynamics of tRNA in free and bound states, respectively. These interactions help in the development of catalytically effective organization of the active site. The A76 end of the ^tttRNA^Ser exhibits fast dynamics in free State, which is significantly slowed down within the active site bound with adenylate. The loops change their conformation via multimodal dynamics (a slow diffusive mode of nanosecond time scale and fast librational mode of dynamics in picosecond time scale). The active site residues of the motif 2 loop approach the proximal bases of tRNA and adenylate by slow diffusive motion (in nanosecond time scale) and make conformational changes of the respective side chains via ultrafast librational motion to develop precise hydrogen bond geometry. Presence of bound Mg²⁺ ions around tRNA and dynamically slow bound water are other common features of both aaRSs. The presence of dynamically rigid Zinc ion coordination sphere and bipartite mode of recognition of ^ectRNA^Thr are observed.     

Identification of the nucleophilic factors and the productive complex for the editing reaction by leucyl-tRNA synthetase

To ensure fidelity of translation, several aminoacyl-tRNA synthetases (aaRSs) possess editing capability to hydrolyse mis-aminoacylated tRNAs. In this report, based on our previously-modelled structure of leucyl-tRNA synthetase (LeuRS) complexed with valyl-tRNA^Leu, further structural modelling has been performed along with molecular dynamics simulations. This enabled the identification of the nucleophile, which is different from that suggested by the crystal structure of the LeuRS • Nva2AA complex. Our results revealed that the 3′ hydroxyl group of A76 acts as a “gate” to regulate the accessibility of the nucleophile; thus, the opening of the gate leads to the productive complex for the reaction.     

Two Complementary Enzymes for Threonylation of tRNA in Crenarchaeota: Crystal Structure of Aeropyrum pernix Threonyl-tRNA Synthetase Lacking a cis-Editing Domain

In protein synthesis, threonyl-tRNA synthetase (ThrRS) must recognize threonine (Thr) from the 20 kinds of amino acids and the cognate tRNA^Thr from different tRNAs in order to generate Thr-tRNA^Thr. In general, an organism possesses one kind of gene corresponding to ThrRS. However, it has been recently found that some organisms have two different genes for ThrRS in the genome, suggesting that their proteins ThrRS-1 and ThrRS-2 function separately and complement each other in the threonylation of tRNA^Thr, one for catalysis and the other for trans-editing of misacylated Ser-tRNA^Thr. In order to clarify their three-dimensional structures, we performed X-ray analyses of two putatively assigned ThrRSs from Aeropyrum pernix (ApThrRS-1 and ApThrRS-2). These proteins were overexpressed in Escherichia coli, purified, and crystallized. The crystal structure of ApThrRS-1 has been successfully determined at 2.3 Å resolution. ApThrRS-1 is a dimeric enzyme composed of two identical subunits, each containing two domains for the catalytic reaction and for anticodon binding. The essential editing domain is completely missing as expected. These structural features reveal that ThrRS-1 catalyzes only the aminoacylation of the cognate tRNA, suggesting the necessity of the second enzyme ThrRS-2 for trans-editing. Since the N-terminal sequence of ApThrRS-2 is similar to the sequence of the editing domain of ThrRS from Pyrococcus abyssi, ApThrRS-2 has been expected to catalyze deaminoacylation of a misacylated serine moiety at the CCA terminus.