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.
The aminoacyl-tRNA synthetases (aaRSs) 2 catalyze the activation of cognate amino acids and their transfer to the 3Ј-end of corresponding tRNA molecules. The aaRSs are a highly conserved family of enzymes comprised of two distinct structural groups referred to as classes I and II (1-3), with a notable exception of LysRS representatives, which belong to both classes (4). Although the catalytic mechanisms of various aaRSs are broadly similar (5), each enzyme has developed a high specificity in recognizing its cognate amino acid and tRNA, which is pivotal for accurate translation of the genetic code (1). The discrimination of the amino acids is based on recognizing the differences in the size and charge of the molecules (6). The specificity of tRNA selection depends on a set of identity determinants that are mostly located at two distal extremities: the anticodon loop and the amino acid accepting stem. In a few instances, identity elements are also found in the D-arm, T-arm, and variable loop. They can either act as positive determinants that enhance aminoacylation or negative ones that prevent aminoacylation. The recognition of tRNAs by synthetases can also be affected by the modification of particular nucleotides (7,8). AaRSs show divergent strategies for tRNA recognition. Most notably, class I and class II aaRSs (including pyrrolysyl-tRNA synthetase, see Ref. 9) approach tRNAs from the minor and major groove sides of the acceptor stem, respectively (10). Although the majority of determinants are in direct contact with cognate synthetases (8), the aminoacylation fidelity is controlled by kinetic differences more than by binding affinities (11).Seryl-tRNA synthetases (SerRSs), which catalyze the aminoacylation of several tRNA Ser isoacceptors and tRNA Sec with serine, can be divided into two structurally different groups: bacterial-type SerRSs function in a variety of archaeal, bacterial, and eukaryotic organisms, whereas the methanogenic-type was found only in methanogenic archaea (12, 13). Furthermore, based on sequence comparison (14, 15) and x-ray analyses, two subgroups of bacterial-type SerRSs were identified: one consists of the enzymes from bacterial sources, best represented by those from Thermus thermophilus (16) and Escherichia coli (3), and an archaeal/eukaryal-type, structurally related to SerRS from archaeon Pyrococcus horikoshii (17).All SerRSs are functional homodimers with a C-terminal active site domain typical for class II aaRSs and an N-terminal domain that is responsible for binding of the long
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.
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