Novel complexes of mammalian translation elongation factor eEF1A·GDP with uncharged tRNA and aminoacyl‐tRNA synthetase

Petrushenko, Zoya M.; Budkevich, Tatyana V.; Shalak, V. F.; Negrutskii, B. S.; El'skaya, A. V.

doi:10.1046/j.1432-1033.2002.03178.x

Cited by 49 publications

(46 citation statements)

References 34 publications

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“…In addition to the lack of specific tRNA recognition capabilities, channeling of aminoacyl-tRNAs is a well documented phenomenon (61,62), and it is unlikely that free-standing charged tRNAs are ever present in the cell and available for interaction with trans-editing domains such as YbaK. Thus, one mechanism that synthetase-like proteins may use to carry out their specific functions in the cell, involves interaction with protein-tRNA complexes.…”

Section: Discussionmentioning

confidence: 99%

Cys-tRNAPro Editing by Haemophilus influenzae YbaK via a Novel Synthetase·YbaK·tRNA Ternary Complex

Musier‐Forsyth

2005

Journal of Biological Chemistry

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Aminoacyl-tRNA synthetases are multidomain enzymes that often possess two activities to ensure translational accuracy. A synthetic active site catalyzes tRNA aminoacylation, while an editing active site hydrolyzes mischarged tRNAs. Prolyl-tRNA synthetases (ProRS) have been shown to misacylate Cys onto tRNA Pro , but lack a Cys-specific editing function. The synthetase-like Haemophilus influenzae YbaK protein was recently shown to hydrolyze misacylated Cys-tRNA Pro in trans. However, the mechanism of specific substrate selection by this single domain hydrolase is unknown. Here, we demonstrate that YbaK alone appears to lack specific tRNA recognition capabilities. Moreover, YbaK cannot compete for aminoacyl-tRNAs in the presence of elongation factor Tu, suggesting that YbaK acts before release of the aminoacyl-tRNA from the synthetase. In support of this idea, cross-linking studies reveal the formation of binary (ProRS⅐YbaK) and ternary (ProRS⅐YbaK⅐tRNA) complexes. The binding constants for the interaction between ProRS and YbaK are 550 nM and 45 nM in the absence and presence of tRNA Pro , respectively. These results suggest that the specificity of trans-editing by YbaK is ensured through formation of a novel ProRS⅐YbaK⅐tRNA complex.Aminoacyl-tRNA synthetases maintain the high fidelity of protein synthesis by activating specific amino acids and transferring them to cognate tRNAs in a process known as aminoacylation. However, some synthetases are known to misactivate noncognate amino acids, transferring them onto their cognate tRNAs. Therefore, to ensure accurate translation of the genetic code, these enzymes, which include members of both classes of synthetases, have evolved proofreading or editing functions (1-17). Pretransfer editing involves hydrolysis of the misactivated aminoacyl-adenylate, whereas post-transfer editing refers to specific hydrolysis of misacylated tRNA.To clear noncognate amino acids, a double-sieve mechanism was proposed, wherein larger amino acids are rejected by the aminoacylation active site (i.e. coarse sieve) while smaller amino acids are cleared in a second editing active site (i.e. fine sieve) (18,19). This hypothesis was confirmed by more recent biochemical and x-ray crystallographic studies (20). Although the exact site of pretransfer editing is still an open question in most systems (21), class I editing enzymes, including isoleucyl-tRNA synthetase, valyl-tRNA synthetase, and leucyl-tRNA synthetase, use the highly conserved connective polypeptide 1 domain to edit misacylated tRNAs (1, 2, 4 -8, 12). In contrast, the post-transfer editing domains used by class II synthetases are much more diverse. The internal editing domain of alanyl-tRNA synthetase (AlaRS) 2 and the N-terminal editing domain of threonyl-tRNA synthetase (with the exception of the archaebacterial enzymes) share sequence similarity (9, 22), while the editing domains of bacterial prolyl-tRNA synthetase (ProRS) (13) and phenylalanyl-tRNA synthetase (17) appear to be unique, sharing no sequence homology to any of the ot...

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Section: Discussionmentioning

confidence: 99%

Cys-tRNAPro Editing by Haemophilus influenzae YbaK via a Novel Synthetase·YbaK·tRNA Ternary Complex

Musier‐Forsyth

2005

Journal of Biological Chemistry

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“…In fact, PhoAlaX and previously characterized trans-editing AlaXs (4) slightly deacylated cognate Ala-tRNA Ala when the concentration of the enzyme and temperature were increased (data not shown). Moreover, it has been shown that charged tRNAs are channeled from aaRSs to ribosomes without releasing free charged tRNAs (28,29). Therefore, the editing should collaborate with aminoacylation, and it is likely that duplication of the editing activities by the free-standing domains is not necessary for the ordinary translation pathway.…”

Section: Discussionmentioning

confidence: 99%

Molecular basis of alanine discrimination in editing site

Sokabe

Okada

Yao

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

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AlaX is the homologue of the class II alanyl-tRNA synthetase editing domain and has been shown to exhibit autonomous editing activity against mischarged tRNA Ala . Here, we present the structures of AlaX from the archaeon Pyrococcus horikoshii in apo form, complexed with zinc, and with noncognate amino acid L-serine and zinc. Together with mutational analysis, we demonstrated that the conserved Thr-30 hydroxyl group located near the ␤-methylene of the bound serine is responsible for the discrimination of noncognate serine from cognate alanine, based on their chemical natures. Furthermore, we confirmed that the conserved Gln-584 in alanyl-tRNA synthetase, which corresponds to Thr-30 of AlaX, is also critical for discrimination. These observations strongly suggested conservation of the chemical discrimination among trans-and cis-editing of tRNA Ala .alanyl-tRNA synthetase ͉ class II tRNA synthetase ͉ crystal structure ͉ trans-editing A minoacyl-tRNA synthetases (aaRSs) establish the genetic code through aminoacylation of cognate tRNA (1). However, in some aaRSs, the affinity difference of the active site is not large enough to distinguish among similar amino acids with sufficient accuracy. Therefore, during evolution, an additional editing domain that specifically hydrolyzes mischarged tRNAs has assembled with the catalytic domain to comprise contemporary aaRSs (2). In accordance with this model, the genes that autonomously encode an editing domain were distributed in many organisms (3-5), and, indeed, some of them are shown to be responsible for the transediting activity of mischarged tRNAs (4, 5). AlaX is the one such protein that shows homology to the class II alanyl-tRNA synthetase (AlaRS) editing domain ( Fig. 1) and is widely scattered among all three kingdoms of life (3). The specific activities of the archaeal AlaXs from Methanosarcina barkeri and Sulfolobus solfataricus have recently been shown to specifically hydrolyze mischarged Ser-and Gly-tRNA Ala in vitro (4).In contrast to the well established class I aaRSs, information regarding editing of the evolutionarily distinct class II aaRSs (to which AlaRS belongs) has only recently begun to emerge. The editing mechanism of the threonyl system has been investigated by structural analyses of the bacterial threonyl-tRNA synthetase (ThrRS) editing domain (hereafter called ThrRS-N2) complexed with the serine product or with the substrate analogue seryl-3Ј-aminoadenosine (SerA76) (6). These analyses showed that (i) cognate threonine and noncognate serine are discriminated by steric exclusion of the additional ␥-methyl group of threonine by the conserved His-77, Tyr-104, and Asp-180, and (ii) the HXXXH and CXXXH motifs characteristic of ThrRS-N2 should not bind a zinc ion for catalysis, despite the capability to bind zinc (7). The AlaX͞AlaRS editing domains are evolutionarily related to ThrRS-N2 and share the characteristic HXXXH and CXXXH motifs (Fig. 1) (4, 8), which were also shown to be important for the deacylation activity of AlaRS (9). The inclusion of non...

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“…While the ternary complex from bacteria is very well-characterized from a biochemical (11, 16 -18) as well as a structural (19,20) point of view, the available information about the corresponding eukaryotic eEF1A⅐GTP⅐aa-tRNA complex is more sparse and divergent. Notably, the existence of non-canonical mammalian eEF1A⅐GDP complexes with deacylated tRNA was suggested (21). The equilibrium dissociation constant, K d , of the latter complex was estimated to 20 nM (21), a value which is comparable to that of canonical EF-Tu⅐GTP⅐aa-tRNA, and 1000 times lower than the K d of the EF-Tu⅐GDP⅐aa-tRNA complex (16).…”

Section: ؊1 Smentioning

confidence: 99%

Kinetics of the Interactions between Yeast Elongation Factors 1A and 1Bα, Guanine Nucleotides, and Aminoacyl-tRNA

Gromadski

Schümmer²,

Strømgaard³

et al. 2007

Journal of Biological Chemistry

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The interactions of elongation factor 1A (eEF1A) from Saccharomyces cerevisiae with elongation factor 1B␣ (eEF1B␣), guanine nucleotides, and aminoacyl-tRNA were studied kinetically by fluorescence stopped-flow. eEF1A has similar affinities for GDP and GTP, 0.4 and 1.1 M, respectively. Dissociation of nucleotides from eEF1A in the absence of the guanine nucleotide exchange factor is slow (about 0.1 s ؊1 ) and is accelerated by eEF1B␣ by 320-fold and 250-fold for GDP and GTP, respectively. The rate constant of eEF1B␣ binding to eEF1A (10 7 -10 8 M ؊1 s ؊1 ) is independent of guanine nucleotides. At the concentrations of nucleotides and factors prevailing in the cell, the overall exchange rate is expected to be in the range of 6 s ؊1

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Novel complexes of mammalian translation elongation factor eEF1A·GDP with uncharged tRNA and aminoacyl‐tRNA synthetase

Cited by 49 publications

References 34 publications

Cys-tRNAPro Editing by Haemophilus influenzae YbaK via a Novel Synthetase·YbaK·tRNA Ternary Complex

Cys-tRNAPro Editing by Haemophilus influenzae YbaK via a Novel Synthetase·YbaK·tRNA Ternary Complex

Molecular basis of alanine discrimination in editing site

Kinetics of the Interactions between Yeast Elongation Factors 1A and 1Bα, Guanine Nucleotides, and Aminoacyl-tRNA

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