Identification of thermodynamically relevant interactions between EF-Tu and backbone elements of tRNA

Pleiss, Jeffrey A.; Uhlenbeck, Olke C.

doi:10.1006/jmbi.2001.4612

Cited by 41 publications

(47 citation statements)

References 45 publications

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“…First, the tightest binding tRNAs tend to have AU and GU base pairs at positions 49-65 and 7-66 at the junction of the acceptor and T stems. Second, the weaker binding tRNAs tend to have a mismatch or AU pair at 1-72 and a GU pair at 50-64, which may prevent the formation of thermodynamically important 2Ј hydroxyl contacts that form at positions 1 and 64 (16). Interestingly, all of these sites have previously been proposed to be ''antideterminants'' that reduce EF-Tu binding to tRNA fMet or tRNA Sec .…”

Section: Discussionmentioning

confidence: 99%

“…First, because the tRNA Phe and tRNA Cys complexes were crystallized in different space groups, it is also possible that the observed differences in the protein-RNA contacts are the result of different packing constraints in the crystal lattice and do not reflect the interaction in solution (19,20). Indeed, several of the 2Ј hydroxyl contacts in the tRNA Phe structure, which differ from those in the tRNA Cys structure, do not contribute to the overall binding energy (16). Thus, not all of the observed differences in the two x-ray structures may be relevant to the specificity in solution.…”

Section: Discussionmentioning

confidence: 99%

“…EF-Tu from T. thermophilus was overexpressed in E. coli and purified as described (16). EF-Tu⅐GTP was prepared immediately before use by incubating 1 M EF-Tu⅐GDP, 3 mM phosphoenolpyruvate, 30 g͞ml pyruvate kinase, 10 mM DTT, 20 M GTP, 20 mM MgCl 2 , 50 mM K-Hepes (pH 7.0), and 0.05-3.5 M NH 4 Cl at 37°C for 3 h (17).…”

Section: Methodsmentioning

confidence: 99%

“…After the addition of 10 l of 0.2 mg͞ml RNase A, 10-l aliquots were removed at various times, quenched into 100 l of 10% trichloroacetic acid (TCA) containing 0.1 mg͞ml of unfractionated tRNA, and filtered through a nitrocellulose membrane. Samples were washed and counted as described (16). Dissociation rates were measured at least three times at each NH 4 Cl concentration, and the mean value of k off was determined.…”

Section: Methodsmentioning

confidence: 99%

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The tRNA Specificity of Thermus thermophilus EF-Tu

Asahara

Uhlenbeck

2002

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

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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...

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

The tRNA Specificity of Thermus thermophilus EF-Tu

Asahara

Uhlenbeck

2002

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

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101

112

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“…Titration curves were plotted from data averaged from three experiments and fit to the following equation (21) by using the graphing program KALEIDAGRAPH (Abelbeck Software, Reading, PA) to estimate Interaction of the C-terminal tails of S9 and S13 with tRNA (red) in the Thermus thermophilus 30S subunit P site (5,6). Large (S9⌬26, S13⌬36) and small (S9⌬3, S13⌬5) tail deletions are red and yellow, respectively; a C-terminal extension of S13 present in T. thermophilus but not in E. coli is gray.…”

Section: Methodsmentioning

confidence: 99%

Creating ribosomes with an all-RNA 30S subunit P site

Hoang

Fredrick

Noller

2004

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

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Ribosome crystal structures have revealed that two small subunit proteins, S9 and S13, have C-terminal tails, which, together with several features of 16S rRNA, contact the anticodon stem-loop of P-site tRNA. To test the functional importance of these protein tails, we created genomic deletions of the C-terminal regions of S9 and S13. All of the tail deletions, including double mutants containing deletions in both S9 and S13, were viable, showing that Escherichia coli cells can synthesize all of their proteins by using ribosomes that contain 30S P sites composed only of RNA. However, these mutants have slower growth rates, indicating that the tails may play a supporting functional role in translation. In vitro analysis shows that 30S subunits purified from the S13 deletion mutants have a generally decreased affinity for tRNA, whereas deletion of the S9 tail selectively affects the binding of tRNAs whose anticodon stem sequences are most divergent from that of initiator tRNA. E xtensive evidence from more than three decades of experiments (1) supports the idea that ribosomal function is based primarily on rRNA (2, 3). This is most clearly seen in ribosome crystal structures, which show that the functional region of the ribosome, at the subunit interface, is composed mostly of rRNA, whereas the ribosomal proteins are found mainly at the periphery of the ribosome (4-7). However, there are a number of instances where ribosomal proteins contact tRNA (6). One of these occurs in the 30S P site, which plays a major role in initiation of protein synthesis, binding the initiator tRNA and positioning the start codon of mRNA. The 30S P site is also responsible for binding and positioning the anticodon stem-loop (ASL) of peptidyl-tRNA during polypeptide elongation and for maintaining the translational reading frame when the A site is vacant. Binding of the ASL depends not only on base-pairing with the P-site codon of mRNA, but also on interactions with the 30S subunit itself. In addition to contacts involving several elements of 16S rRNA, the extended C-terminal tails of two proteins, S9 and S13, penetrate the 30S P site within contact distance of the ASL (5, 6). The universally conserved C-terminal arginine of S9 appears to contact phosphate 35 at the apex of the anticodon loop of the P-site tRNA (Fig. 1). The C-terminal tail of S9 is phylogenetically invariant in length, an observation that is explained by the crystal structure, which shows that the P-site tRNA would physically block extension of the S9 tail. The tail of S13 runs parallel to the anticodon stem, crossing the tRNA backbone between positions 29 and 30 and is thus more tolerant of variations in length (Fig. 1). Although more variable in sequence, the S13 tail always contains several basic side chains, which can make electrostatic interactions with tRNA phosphates. The interactions of the S9 and S13 protein tails with P-site tRNA and their phylogenetic conservation suggest that they are directly involved in the function of the 30S P site.Early in vitro reconsti...

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Structure of the Ribosome

Blaha¹,

Иванов²

2004

Protein Synthesis and Ribosome Structure

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Identification of thermodynamically relevant interactions between EF-Tu and backbone elements of tRNA

Cited by 41 publications

References 45 publications

The tRNA Specificity of Thermus thermophilus EF-Tu

The tRNA Specificity of Thermus thermophilus EF-Tu

Creating ribosomes with an all-RNA 30S subunit P site

Structure of the Ribosome

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