PRC1-labeled microtubule bundles and kinetochore pairs show one-to-one association in metaphase.

In the mitotic spindle, kinetochore microtubules form k-fibers, whereas overlap or interpolar microtubules form antiparallel arrays containing the cross-linker protein regulator of cytokinesis 1 (PRC1). We have recently shown that an overlap bundle, termed bridging fiber, links outermost sister k-fibers. However, the relationship between overlap bundles and k-fibers throughout the spindle remained unknown. Here, we show that in a metaphase spindle more than 90% of overlap bundles act as a bridge between sister k-fibers. We found that the number of PRC1-GFP-labeled bundles per spindle is nearly the same as the number of kinetochore pairs. Live-cell imaging revealed that kinetochore movement in the equatorial plane of the spindle is highly correlated with the movement of the coupled PRC1-GFP-labeled fiber, whereas the correlation with other fibers decreases with increasing distance. Analysis of endogenous PRC1 localization confirmed the results obtained with PRC1-GFP PRC1 knockdown reduced the bridging fiber thickness and interkinetochore distance throughout the spindle, suggesting a function of PRC1 in bridging microtubule organization and force balance in the metaphase spindle.

A single amino acid substitution affects the substrate specificity of the seryl-tRNA synthetase homologue.

Recently described and characterized Bradyrhizobium japonicum glycine:[carrier protein] ligase 1 (Bj Gly:CP ligase 1), a homologue of methanogenic type seryl-tRNA synthetase (SerRS) is an intriguing enzyme whose physiological role is not yet known. While aminoacyl-tRNA synthetases supply ribosome with amino acids for protein biosynthesis, this homologue transfers the activated amino acid to a specific carrier protein. Despite remarkable structural similarity between the Bj Gly:CP ligase 1 and the catalytic core domain of methanogenic type SerRS, the ligase displays altered and relaxed substrate specificity. In contrast to methanogenic SerRS which exclusively activates serine, the Bj Gly:CP ligase 1 predominantly activates glycine. Besides, it shows low activity in the presence of alanine, but it is incapable of activating serine. The detailed computational study aiming to address this unexpected substrate specificity toward the small aliphatic amino acids revealed the A281G Bj Gly:CP ligase 1 mutant as the most promising candidate with reconstituted catalytic activity toward the larger substrates. The A281G mutation is predicted to increase the active site volume, allowing alanine and serine to establish important hydrogen bonds within the active site, and to adopt an optimal orientation for the reaction. The results were tested by the site-directed mutagenesis experiments coupled with in vitro kinetic assays. It was found that the A281G substitution greatly affects the enzyme specificity and allows efficient activation of both polar and small aliphatic amino acids (serine, glycine and alanine), confirming predictions and conclusions based on molecular dynamics simulations.

Substrate recognition and fidelity of maize seryl-tRNA synthetases.

Aminoacyl-tRNA synthetases (aaRSs) catalyze the attachment of amino acids to their cognate tRNAs to establish the genetic code. To obtain the high degree of accuracy that is observed in translation, these enzymes must exhibit strict substrate specificity for their cognate amino acids and tRNAs. We studied the requirements for tRNA(Ser) recognition by maize cytosolic seryl-tRNA synthetase (SerRS). The enzyme efficiently recognized bacterial and eukaryotic tRNAs(Ser) indicating that it can accommodate various types of tRNA(Ser) structures. Discriminator base G73 is crucial for recognition by cytosolic SerRS. Although cytosolic SerRS efficiently recognized bacterial tRNAs(Ser), it is localized exclusively in the cytosol. The fidelity of maize cytosolic and dually targeted organellar SerRS with respect to amino acid recognition was compared. Organellar SerRS exhibited higher discrimination against tested non-cognate substrates as compared with cytosolic counterpart. Both enzymes showed pre-transfer editing activity implying their high overall accuracy. The contribution of various reaction pathways in the pre-transfer editing reactions by maize enzymes were different and dependent on the non-cognate substrate. The fidelity mechanisms of maize organellar SerRS, high discriminatory power and proofreading, indicate that aaRSs in general may play an important role in translational quality control in plant mitochondria and chloroplasts.

Identification of amino acids in the N-terminal domain of atypical methanogenic-type Seryl-tRNA synthetase critical for tRNA recognition.

Seryl-tRNA synthetase (SerRS) from methanogenic archaeon Methanosarcina barkeri, contains an idiosyncratic N-terminal domain, composed of an antiparallel beta-sheet capped by a helical bundle, connected to the catalytic core by a short linker peptide. It is very different from the coiled-coil tRNA binding domain in bacterial-type SerRS. Because the crystal structure of the methanogenic-type SerRSxtRNA complex has not been obtained, a docking model was produced, which indicated that highly conserved helices H2 and H3 of the N-terminal domain may be important for recognition of the extra arm of tRNA(Ser). Based on structural information and the docking model, we have mutated various positions within the N-terminal region and probed their involvement in tRNA binding and serylation. Total loss of activity and inability of the R76A variant to form the complex with cognate tRNA identifies Arg(76) located in helix H2 as a crucial tRNA-interacting residue. Alteration of Lys(79) positioned in helix H2 and Arg(94) in the loop between helix H2 and beta-strand A4 have a pronounced effect on SerRSxtRNA(Ser) complex formation and dissociation constants (K(D)) determined by surface plasmon resonance. The replacement of residues Arg(38) (located in the loop between helix H1 and beta-strand A2), Lys(141) and Asn(142) (from H3), and Arg(143) (between H3 and H4) moderately affect both the serylation activity and the K(D) values. Furthermore, we have obtained a striking correlation between these results and in vivo effects of these mutations by quantifying the efficiency of suppression of bacterial amber mutations, after coexpression of the genes for M. barkeri suppressor tRNA(Ser) and a set of mMbSerRS variants in Escherichia coli.

Recognition between tRNASer and archaeal seryl-tRNA synthetases monitored by suppression of bacterial amber mutations.

Two dissimilar seryl-tRNA synthetases (SerRSs) exist in Methanosarcina barkeri: one of bacterial type (bMbSerRS) and the other resembling SerRSs present only in methanogenic archaea (mMbSerRS). While the expression of the archaeal bMbSerRS gene in Escherichia coli complements the function of thermolabile SerRS at a nonpermissive temperature, mMbSerRS does not. Our recent X-ray structural analysis of mMbSerRS revealed an idiosyncratic N-terminal domain and a catalytic zinc ion in the active site, identifying methanogenic-type SerRSs as atypical members of the SerRS family. To shed further light on substrate discrimination by methanogenic-type SerRS, we developed an in vivo system in E. coli to study tRNA serylation by mMbSerRS variants. We show that coexpression of the M. barkeri SerRS gene, encoding either bacterial- or methanogenic-type SerRS, with the gene for cognate archaeal suppressor tRNA leads to suppression of bacterial amber mutations, implying that the E. coli translation machinery can use serylated tRNA from methanogenic archaea as a substrate in protein synthesis. Furthermore, because serylation of M. barkeri serine-specific tRNA by endogenous E. coli SerRS is negligible, suppression is entirely dependent on recognition between archaeal partners (mMbSerRS/suppressor tRNA(Ser)). Thus, the efficiency of suppression by mMbSerRS variants quantified in the described beta-galactosidase-based reporter system, accurately reflects enzymes' serylation propensity obtained by in vitro kinetic measurements.

Maize seryl-tRNA synthetase: specificity of substrate recognition by the organellar enzyme.

In our study of seryl-tRNA formation in maize, we investigated the enzymes involved in serylation. Only two dissimilar seryl-tRNA synthetase (SerRS) cDNA clones were identified in the Zea mays EST (expressed sequence tag) databases. One encodes a seryl-tRNA synthetase, which presumably functions in the organelles (SerZMm), while the other synthetase product is more similar to eukaryotic cytosolic counterparts (SerZMc). The expression of SerZMm in Saccharomyces cerevisiae resulted in complementation of mutant respiratory phenotype, caused by a disruption of the nuclear gene, presumably encoding yeast mitochondrial seryl-tRNA synthetase (SerSCm). Purified mature SerZMm displays tRNA-assisted serine activation and aminoacylates maize mitochondrial and chloroplast tRNA(Ser) transcripts with similar efficiencies, raising the possibility that only two isoforms of seryl-tRNA synthetase may be sufficient to catalyze seryl-tRNA(Ser) formation in three cellular compartments of Zea mays. Phylogenetic analysis suggests that SerZMm is of mitochondrial origin.