Discrimination against misacylated tRNA by chloroplast elongation factor Tu

100

112

By introducing a GAC anticodon, 21 different Escherichia coli tRNAs were misacylated with either phenylalanine or valine and assayed for their affinity to Thermus thermophilus elongation factor Tu (EF-Tu)⅐GTP by using a ribonuclease protection assay. The presence of a common esterified amino acid permits the thermodynamic contribution of each tRNA body to the overall affinity to be evaluated. The E. coli elongator tRNAs exhibit a wide range of binding affinities that varied from ؊11.7 kcal͞mol for Val-tRNA Glu to ؊8.1 kcal͞mol for Val-tRNA Tyr , clearly establishing EF-Tu⅐GTP as a sequence-specific RNA-binding protein. Because the ionic strength dependence of koff varied among tRNAs, some of the affinity differences are the results of a different number of phosphate contacts formed between tRNA and protein. Because EF-Tu is known to contact only the phosphodiester backbone of tRNA, the observed specificity must be a consequence of an indirect readout mechanism. E longation factor Tu (EF-Tu) binds GTP and aminoacyl tRNA (aa-tRNA) to form a ternary complex that subsequently binds ribosome and participates in codon-directed binding of the aa-tRNA to the ribosomal A site. Although EFTu⅐GTP binds poorly to tRNAs lacking the esterified amino acid (1), the protein is generally considered to lack specificity because it binds all elongator aa-tRNAs with a similar affinity (2-4). However, recent experiments have shown that EF-Tu⅐GTP exhibits substantial specificity for both the esterified amino acid and the tRNA body of aa-tRNAs (5). This specificity was previously unappreciated because the contributions of the amino acid and the tRNA body to the overall affinity are arranged in a compensatory manner such that the cognate aa-tRNAs all bind with similar affinities. However, misacylated tRNAs were found to bind EF-Tu⅐GTP with a wide range of affinities that were either tighter or weaker than the cognate aa-tRNAs (5). The four different tRNA bodies that were tested displayed about a 100-fold range of K D values with Thermus thermophilus EFTu⅐GTP when each was esterified with the same amino acid. The same range of K D values was observed when the four tRNAs were esterified with a different common amino acid, clearly establishing that EF-Tu shows specificity toward the tRNA body. However, the four tRNAs used in these experiments (Escherichia coli tRNA Ala , tRNA Gln , tRNA Val , and yeast tRNA Phe ) have very similar overall architectures, raising the possibility that EFTu⅐GTP could exhibit a much larger range of affinities with tRNAs of different architectures.Experiments presented here take advantage of the important role of the anticodon as a identity element for valyl-tRNA synthetase (ValRS) and phenylalanyl-tRNA synthetase (PheRS) (6-10) to prepare 21 different E. coli tRNAs that were misacylated with either valine or phenylalanine. By comparing the affinities of the different tRNAs esterified with a common amino acid, the specificity of EF-Tu⅐GTP for the different tRNA bodies was established. Materials and MethodsE. coli...

Section: Discussionmentioning

confidence: 64%

The tRNA Specificity of Thermus thermophilus EF-Tu

Asahara

Uhlenbeck

2002

100

112

“…Utilization of Glu-tRNA Gln in protein synthesis would cause lethality. However, it was shown in the chloroplast system that EF-Tu rejects Glu-tRNA Gln (7) so that the misacylated tRNA is not brought to the ribosome. It is interesting that EF-Tu discriminates against the only aminoacylated tRNA that is a substrate for Glu-AdT.…”

Section: Discussionmentioning

confidence: 99%

“…3). The mischarged tRNA may be excluded from protein synthesis because of poor recognition by elongation factor (EF)-Tu, as is the case in chloroplasts (7). In the presence of glutamine as amine donor and ATP, GlutRNA Gln is then transamidated by Glu-tRNA Gln amidotransferase (Glu-AdT), presumably through an activated ␥-phosphoGlu-tRNA Gln intermediate (8).…”

mentioning

confidence: 99%

Glu-tRNA ^Gln amidotransferase: A novel heterotrimeric enzyme required for correct decoding of glutamine codons during translation

Curnow

Hong

Yuan

et al. 1997

301

310

The three genes, gatC, gatA, and gatB, which constitute the transcriptional unit of the Bacillus subtilis glutamyl-tRNA Gln amidotransferase have been cloned. Expression of this transcriptional unit results in the production of a heterotrimeric protein that has been purified to homogeneity. The enzyme furnishes a means for formation of correctly charged Gln-tRNA Gln through the transamidation of misacylated Glu-tRNA Gln , functionally replacing the lack of glutaminyl-tRNA synthetase activity in Gram-positive eubacteria, cyanobacteria, Archaea, and organelles. Disruption of this operon is lethal. This demonstrates that transamidation is the only pathway to Gln-tRNA Gln in B. subtilis and that glutamyl-tRNA Gln amidotransferase is a novel and essential component of the translational apparatus.

“…We were able to successfully reconstitute Gln-tRNA Gln formation in vitro using the recombinant mtGluRS and hGatCAB. (25). Thus, we hypothesized that a similar mechanism may maintain the fidelity of translation in human mitochondria.…”

Section: In Vitro Reconstitution Of Amidotransfer Reaction By Hgatcabmentioning

confidence: 99%

Biogenesis of glutaminyl-mt tRNA ^Gln in human mitochondria

Nagao

Suzuki²,

Katoh³

et al. 2009

Mammalian mitochondrial (mt) tRNAs, which are required for mitochondrial protein synthesis, are all encoded in the mitochondrial genome, while mt aminoacyl-tRNA synthetases (aaRSs) are encoded in the nuclear genome. However, no mitochondrial homolog of glutaminyl-tRNA synthetase (GlnRS) has been identified in mammalian genomes, implying that Gln-tRNA Gln is synthesized via an indirect pathway in the mammalian mitochondria. We demonstrate here that human mt glutamyl-tRNA synthetase (mtGluRS) efficiently misaminoacylates mt tRNA Gln to form Glu-tRNA Gln . In addition, we have identified a human homolog of the Glu-tRNA Gln amidotransferase, the hGatCAB heterotrimer. When any of the hGatCAB subunits were inactivated by siRNA-mediated knock down in human cells, the Glu-charged form of tRNA Gln accumulated and defects in respiration could be observed. We successfully reconstituted in vitro Gln-tRNA Gln formation catalyzed by the recombinant mtGluRS and hGatCAB. The misaminoacylated form of tRNA Gln has a weak binding affinity to the mt elongation factor Tu (mtEF-Tu), indicating that the misaminoacylated form of tRNA Gln is rejected from the translational apparatus to maintain the accuracy of mitochondrial protein synthesis.EF-Tu ͉ Glu-tRNAGln amidotransferase ͉ glutamyl-tRNA synthetase ͉ mitochondrial tRNA