The inactive X chromosome (Xi) serves as a model to understand gene silencing on a global scale. Here, we perform “identification of direct RNA interacting proteins” (iDRiP) to isolate a comprehensive protein interactome for Xist, an RNA required for Xi silencing. We discover multiple classes of interactors, including cohesins, condensins, topoisomerases, RNA helicases, chromatin remodelers and modifiers, which synergistically repress Xi transcription. Inhibiting two or three interactors destabilizes silencing. While Xist attracts some interactors, it repels architectural factors. Xist evicts cohesins from the Xi and directs an Xi-specific chromosome conformation. Upon deleting Xist, the Xi acquires the cohesin-binding and chromosomal architecture of the active X. Our study unveils many layers of Xi repression and demonstrates a central role for RNA in the topological organization of mammalian chromosomes.

Aminoacyl-tRNA synthetases (aaRSs) join amino acids to 1 of 2 terminal hydroxyl groups of their cognate tRNAs, thereby contributing to the overall fidelity of protein synthesis. In class II histidyltRNA synthetase (HisRS) the nonbridging Sp-oxygen of the adenylate is a potential general base for aminoacyl transfer. To test for conservation of this mechanism in other aaRSs and the role of terminal hydroxyls of tRNA in aminoacyl transfer, we investigated the class II Escherichia coli threonyl-tRNA synthetase (ThrRS). As with other class II aaRSs, the rate-determining step for ThrRS is amino acid activation. In ThrRS, however, the 2-OH of A76 of tRNA Thr and a conserved active-site histidine (His-309) collaborate to catalyze aminoacyl transfer by a mechanism distinct from HisRS. Conserved residues in the ThrRS active site were replaced with alanine, and then the resulting mutant proteins were analyzed by steady-state and rapid kinetics. Nearly all mutants preferentially affected the amino acid activation step, with only a modest effect on aminoacyl transfer. By contrast, H309A ThrRS decreased transfer 242-fold and imposed a kinetic block to CCA accommodation. His-309 hydrogen bonds to the 2-OH of A76, and substitution of the latter by hydrogen or fluorine decreased aminoacyl transfer by 763-and 94-fold, respectively. The proton relay mechanism suggested by these data to promote aminoacylation is reminiscent of the NAD ؉ -dependent mechanisms of alcohol dehydrogenases and sirtuins and the RNA-mediated catalysis of the ribosomal peptidyl transferase center.proton relay ͉ threonine ͉ translation ͉ aminoacyl-tRNA synthetase ͉ transient kinetics T he attachment of specific amino acids to their cognate tRNAs by aminoacyl tRNA synthetases (aaRSs) enables ribosomes to assemble amino acids into proteins accurately, as dictated by the sequence of codons in the mRNA. Two distinct classes of aaRSs catalyze aminoacylation, which occurs in 2 steps. During amino acid activation, the cognate amino acid is condensed with ATP to form an enzyme-bound adenylate, with release of pyrophosphate. This is followed by aminoacyl transfer, where the amino acid is transferred to either the 2Ј (class I aaRSs) or 3Ј (class II aaRSs) hydroxyl of A76 to generate aminoacylated tRNA, along with AMP (1, 2).The active sites of aaRSs from both classes are remarkably devoid of candidate residues to serve as general bases for aminoacyl transfer. On the basis of structural information in the GlnRS system (3, 4) and later rapid kinetics studies using histidyl-tRNA synthetase (HisRS), the pro-S nonbridging oxygen of the adenylate was proposed to serve as a general base for aminoacyl transfer (5). The latter study also highlighted a role for tRNA in modulating amino acid activation, marking it as the putative rate-determining step for overall aminoacylation. The generality of these proposals has not yet been investigated in detail, even among aaRSs in the same class.Among class II aaRSs, threonyl-tRNA synthetase (ThrRS) shares the canonical class IIa catalyt...

Aminoacyl-tRNA synthetases hydrolyze aminoacyl adenylates and aminoacyl-tRNAs formed from near-cognate amino acids, thereby increasing translational fidelity. The contributions of pre-and post-transfer editing pathways to the fidelity of Escherichia coli threonyl-tRNA synthetase (ThrRS) were investigated by rapid kinetics. In the pre-steady state, asymmetric activation of cognate threonine and noncognate serine was observed in the active sites of dimeric ThrRS, with similar rates of activation. In the absence of tRNA, seryl-adenylate was hydrolyzed 29-fold faster by the ThrRS catalytic domain than threonyl-adenylate. The rate of seryl transfer to cognate tRNA was only 2-fold slower than threonine. Experiments comparing the rate of ATP consumption to the rate of aminoacyl-tRNA AA formation demonstrated that pre-transfer hydrolysis contributes to proofreading only when the rate of transfer is slowed significantly. Thus, the relative contributions of pre-and posttransfer editing in ThrRS are subject to modulation by the rate of aminoacyl transfer.The accurate transfer of genetic information in living systems requires multiple mechanisms to enhance and preserve the fidelity of gene expression, particularly in protein synthesis. The 1-3 errors per 10,000 amino acids incorporated during protein synthesis largely originate from errors in decoding (1) but can arise from previous steps, including the aminoacylation reaction catalyzed by aminoacyl-tRNA synthetases (ARSs) 2 (2). In the first of two half-reactions, ARSs condense amino acid and ATP to form a noncovalently enzyme-bound adenylate, followed by a transfer reaction that produces aminoacyl-tRNA and AMP as products. This highly accurate reaction (on the order of 1 error in 10 5 ) reflects the ability of ARSs to carefully discriminate among chemically similar standard and nonstandard amino acids (3). Mutations in ARSs or their cognate tRNAs can nonetheless create errors associated with significant molecular pathologies, particularly in neural tissues (4).Amino acids that differ by a single methyl group pose a particular discrimination challenge for ARSs (3,5,6). An additional methyl group on an amino acid typically provides no more than ϳ1 kcal/mol of incremental binding energy, imposing an upper limit of discrimination on the order of 1 in 5 (7). To account for the enhanced discrimination seen in living systems, a "double sieve" model was proposed (8). Amino acids larger than cognate are excluded by the "coarse sieve" of the aminoacylation site, whereas smaller or isosteric amino acids are cleaved from the end of the tRNA by the "fine sieve" of the editing site. In principle, noncognate amino acids can be edited both by increased hydrolysis of the adenylate (pre-transfer editing) or by hydrolysis of the mis-acylated tRNA (post-transfer editing). Either or both reactions can formally occur in a dedicated editing site structurally distinct from the standard synthetic site. Because of differences between systems, and the absence of a comprehensive kinetic description ...

During protein synthesis, tRNA serves as the intermediary between cognate amino acids and their corresponding RNA trinucleotide codons. Aminoacyl-tRNA is also a biosynthetic precursor and amino acid donor for other macromolecules. AA-tRNAs allow transformations of acidic amino acids into their amide-containing counterparts, and seryl-tRNASer donates serine for antibiotic synthesis. Aminoacyl-tRNA is also used to cross-link peptidoglycan, to lysinylate the lipid bilayer, and to allow proteolytic turnover via the N-end rule. These alternative functions may signal the use of RNA in early evolution as both a biological scaffold and a catalyst to achieve a wide variety of chemical transformations.

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In all living systems, the fidelity of translation is maintained in part by the editing mechanisms of aminoacyl-tRNA synthetases (ARSs). Some non-proteogenic amino acids, including β-hydroxynorvaline (HNV) are nevertheless efficiently aminoacylated and become incorporated into proteins. To investigate the basis of HNV's ability to function in protein synthesis, the utilization of HNV by E. coli threonyl-tRNA synthetase (ThrRS) was investigated through both in vitro functional experiments and bacterial growth studies. The measured specificity constant (k cat /K M ) for HNV was found to be only 20-30 fold less than that of cognate threonine. The rate of aminoacyl transfer (10.4 s −1 ) was 10-fold higher than the multiple turnover k cat value (1 s −1 ), indicating that, as for cognate threonine, amino acid activation is likely to be the rate-limiting step. Like non-cognate serine, HNV enhances the ATPase function of the synthetic site, at a rate not increased by non-aminoacylatable (3′-dA76) tRNA. ThrRS also failed to exhibit post-transfer editing activity against HNV. In growing bacteria, the addition of HNV dramatically suppressed growth rates, which indicates either negative phenotypic consequences associated with its incorporation into protein, or inhibition of an unidentified metabolic reaction. The inability of wild ThrRS to prevent utilization of HNV as a substrate illustrates that, for at least one ARS, the naturally occurring enzyme lacks the capability to effectively discriminate against non-proteogenic amino acids that are not encountered under normal physiological conditions. Other examples of 'fidelity escape' in the ARSs may serve as useful starting points in the design of ARSs with specificity for unnatural amino acids.Aminoacyl-tRNA synthetases establish the fidelity of the genetic code in a two-step aminoacylation reaction, covalently linking specific amino acids to their corresponding tRNAs. In the first step, amino acid is condensed with ATP to form the aminoacyl adenylate (adenylation), while in the second step, the amino acid moiety of adenylate is transferred to the A76 of tRNA (aminoacyl transfer), along with the release of pyrophosphate (PPi) and AMP, respectively.