Insights into substrate promiscuity of human seryl-tRNA synthetase.

Seryl-tRNA synthetase (SerRS) attaches L-serine to the cognate serine tRNA (tRNASer) and the noncognate selenocysteine tRNA (tRNASec). The latter activity initiates the anabolic cycle of selenocysteine (Sec), proper decoding of an in-frame Sec UGA codon, and synthesis of selenoproteins across all domains of life. While the accuracy of SerRS is important for overall proteome integrity, it is its substrate promiscuity that is vital for the integrity of the selenoproteome. This raises a question as to what elements in the two tRNA species, harboring different anticodon sequences and adopting distinct folds, facilitate aminoacylation by a common aminoacyl-tRNA synthetase. We sought to answer this question by analyzing the ability of human cytosolic SerRS to bind and act on tRNASer, tRNASec, and 10 mutant and chimeric constructs in which elements of tRNASer were transposed onto tRNASec We show that human SerRS only subtly prefers tRNASer to tRNASec, and that discrimination occurs at the level of the serylation reaction. Surprisingly, the tRNA mutants predicted to adopt either the 7/5 or 8/5 fold are poor SerRS substrates. In contrast, shortening of the acceptor arm of tRNASec by a single base pair yields an improved SerRS substrate that adopts an 8/4 fold. We suggest that an optimal tertiary arrangement of structural elements within tRNASec and tRNASer dictate their utility for serylation. We also speculate that the extended acceptor-TΨC arm of tRNASec evolved as a compromise for productive binding to SerRS while remaining the major recognition element for other enzymes involved in Sec and selenoprotein synthesis.

The crystal structure of yeast mitochondrial ThrRS in complex with the canonical threonine tRNA.

In mitochondria of Saccharomyces cerevisiae, a single aminoacyl-tRNA synthetase (aaRS), MST1, aminoacylates two isoacceptor tRNAs, tRNA1(Thr) and tRNA2(Thr), that harbor anticodon loops of different size and sequence. As a result of this promiscuity, reassignment of the CUN codon box from leucine to threonine is facilitated. However, the mechanism by which a single aaRS binds distinct anticodon loops with high specificity is not well understood. Herein, we present the crystal structure of MST1 in complex with the canonical tRNA2(Thr) and non-hydrolyzable analog of threonyl adenylate. Our structure reveals that the dimeric arrangement of MST1 is essential for binding the 5'-phosphate, the second base pair of the acceptor stem, the first two base pairs of the anticodon stem and the first nucleotide of the variable arm. Further, in contrast to the bacterial ortholog that 'reads' the entire anticodon sequence, MST1 recognizes bases in the second and third position and the nucleotide upstream of the anticodon sequence. We speculate that a flexible loop linking strands ß4 and ß5 may be allosteric regulator that establishes cross-subunit communication between the aminoacylation and tRNA-binding sites. We also propose that structural features of the anticodon-binding domain in MST1 permit binding of the enlarged anticodon loop of tRNA1(Thr).

Depletion of alveolar macrophages ameliorates virus-induced disease following a pulmonary coronavirus infection.

Coronaviruses cause respiratory disease in humans that can range from mild to severe. However, the pathogenesis of pulmonary coronavirus infections is poorly understood. Mouse hepatitis virus type 1 (MHV-1) is a group 2 coronavirus capable of causing severe morbidity and mortality in highly susceptible C3H/HeJ mice. We have previously shown that both CD4 and CD8 T cells play a critical role in mediating MHV-1-induced disease. Here we evaluated the role of alveolar macrophages (AM) in modulating the adaptive immune response and subsequent disease. Depletion of AM using clodronate liposomes administered prior to MHV-1 infection was associated with a significant amelioration of MHV-1-induced morbidity and mortality. AM depletion resulted in a decreased number of virus-specific CD4 T cells in the lung airways. In addition, a significant increase in the frequency and total number of Tregs in the lung tissue and lung airways was observed following MHV-1 infection in mice depleted of AM. Our results indicate that AM play a critical role in modulating MHV-1-induced morbidity and mortality.

The synthesis of methylated, phosphorylated, and phosphonated 3'-aminoacyl-tRNA(Sec) mimics.

The twenty first amino acid, selenocysteine (Sec), is the only amino acid that is synthesized on its cognate transfer RNA (tRNA(Sec)) in all domains of life. The multistep pathway involves O-phosphoseryl-tRNA:selenocysteinyl-tRNA synthase (SepSecS), an enzyme that catalyzes the terminal chemical reaction during which the phosphoseryl-tRNA(Sec) intermediate is converted into selenocysteinyl-tRNA(Sec). The SepSecS architecture and the mode of tRNA(Sec) recognition have been recently determined at atomic resolution. The crystal structure provided valuable insights that gave rise to mechanistic proposals that could not be validated because of the lack of appropriate molecular probes. To further improve our understanding of the mechanism of the biosynthesis of selenocysteine in general and the mechanism of SepSecS in particular, stable tRNA(Sec) substrates carrying aminoacyl moieties that mimic particular reaction intermediates are needed. Here, we report on the accurate synthesis of methylated, phosphorylated, and phosphonated serinyl-derived tRNA(Sec) mimics that contain a hydrolysis-resistant ribose 3'-amide linkage instead of the natural ester bond. The procedures introduced allow for efficient site-specific methylation and/or phosphorylation directly on the solid support utilized in the automated RNA synthesis. For the preparation of (S)-2-amino-4-phosphonobutyric acid-oligoribonucleotide conjugates, a separate solid support was generated. Furthermore, we developed a three-strand enzymatic ligation protocol to obtain the corresponding full-length tRNA(Sec) derivatives. Finally, we developed an electrophoretic mobility shift assay (EMSA) for rapid, qualitative characterization of the SepSecS-tRNA interactions. The novel tRNA(Sec) mimics are promising candidates for further elucidation of the biosynthesis of selenocysteine by X-ray crystallography and other biochemical approaches, and could be attractive for similar studies on other tRNA-dependent enzymes.