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1.
Previous work established that seven-base-pair hairpin microhelices with sequences based on the acceptor stems of alanine, glycine, methionine, and histidine tRNAs can be aminoacylated specifically with their cognate amino acids. To obtain "minimalist" substrates with fewer base pairs, we took advantage of the high thermodynamic stability of RNA tetraloop motifs that are found in ribosomal RNAs. We show here that rationally designed RNA tetraloops with as few as four base pairs are substrates for aminoacylation. Major nucleotide determinants for recognition by the class II synthetases were incorporated into each of the respective tetraloop substrates, resulting in specific aminoacylation by the alanine, glycine, and histidine tRNA synthetases. An analysis of the kinetics of aminoacylation shows that, for the alanine system, the majority of the transition-state stabilization provided by the synthetase-tRNA interaction is reproduced by the interaction of the synthetase with nucleotides in its minimalist tetraloop substrate. In an extension of this work, we also observed specific aminoacylation with the class I methionine tRNA synthetase of RNA tetraloops based on sequences in the acceptor stem of methionine tRNA. Thus, the results demonstrate four different examples where specific aminoacylation is directed by sequences/structures contained in less than half of a turn of an RNA helix. 相似文献
2.
Functional analysis of peptide motif for RNA microhelix binding suggests new family of RNA-binding domains. 总被引:2,自引:0,他引:2
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RNA microhelices that recreate the acceptor stems of transfer RNAs are charged with specific amino acids. Here we identify a two-helix pair in alanyl-tRNA synthetase that is required for RNA microhelix binding. A single point mutation at an absolutely conserved residue in this motif selectively disrupts RNA binding without perturbation of the catalytic site. These results, and findings of similar motifs in the proximity of the active sites of other tRNA synthetases, suggest that two-helix pairs are widespread and provide a structural framework important for contacts with bound RNA substrates. 相似文献
3.
《Critical reviews in biochemistry and molecular biology》2013,48(4):309-322
AbstractThe genetic code is based on the aminoacylation of tRNA with amino acids catalyzed by the aminoacyl-tRNA synthetases. The synthetases are constructed from discrete domains and all synthetases possess a core catalytic domain that catalyzes amino acid activation, binds the acceptor stem of tRNA, and transfers the amino acid to tRNA. Fused to the core domain are additional domains that mediate RNA interactions distal to the acceptor stem. Several synthetases catalyze the aminoacylation of RNA oligonucleotide substrates that recreate only the tRNA acceptor stems. In one case, a relatively small catalytic domain catalyzes the aminoacylation of these substrates independent of the rest of the protein. Thus, the active site domain may represent a primordial synthetase in which polypeptide insertions that mediate RNA acceptor stem interactions are tightly integrated with determinants for aminoacyl adenylate synthesis. The relationship between nucleotide sequences in small RNA oligonucleotides and the specific amino acids that are attached to these oligonucleotides could constitute a second genetic code. 相似文献
4.
RNA minihelices and the decoding of genetic information 总被引:1,自引:0,他引:1
P Schimmel 《FASEB journal》1991,5(8):2180-2187
The rules of the genetic code are determined by the specific aminoacylation of transfer RNAs by aminoacyl transfer RNA synthetase. A straightforward analysis shows that a system of synthetase-tRNA interactions that relies on anticodons for specificity could, in principle, enable most synthetases to distinguish their cognate tRNA isoacceptors from all others. Although the anticodons of some tRNAs are recognition sites for the cognate aminoacyl tRNA synthetases, for other synthetases the anticodon is dispensable for specific aminoacylation. In particular, alanine and histidine tRNA synthetases aminoacylate small RNA minihelices that reconstruct the part of their cognate tRNAs that is proximate to the amino acid attachment site. Helices with as few as six base pairs can be efficiently aminoacylated. The specificity of aminoacylation is determined by a few nucleotides and can be converted from one amino acid to another by the change of only a few nucleotides. These findings suggest that, for a subgroup of the synthetases, there is a distinct code in the acceptor helix of transfer RNAs that determines aminoacylation specificity. 相似文献
5.
An operational RNA code for amino acids and variations in critical nucleotide sequences in evolution
Paul Schimmel 《Journal of molecular evolution》1995,40(5):531-536
An operational RNA code relates specific amino acids to sequences/structures in RNA hairpin helices which reconstruct the seven-base-pair acceptor stems of transfer RNAs. These RNA oligonucleotides are aminoacylated by aminoacyl tRNA synthetases. The specificity and efficiency of aminoacylation are generally determined by three or four nucleotides which are near the site of amino acid attachment. These specificity-determining nucleotides include the so-called discriminator base and one or two base pairs within the first four base pairs of the helix. With three examples considered here, nucleotide sequence variations between the eubacterial E. coli tRNA acceptor stems and their human cytoplasmic and mitochondrial counterparts are shown to include changes of some of the nucleotides known to be essential for aminoacylation by the cognate E. coli enzymes. If the general locations of the specificity-determining nucleotides are the same in E. coli and human RNAs, these RNA sequence variations imply a similar covariation in sequences/structures of the E. coli and human tRNA synthetases. These covariations would reflect the integral relationship between the operational RNA code and the design and evolution of tRNA synthetases.Based on part of a presentation made at a workshop- Aminoacyl-tRNA Synthetases and the Evolution of the Genetic Code-held at Berkeley, CA, July 17–20, 1994 相似文献
6.
7.
Evidence for a conserved relationship between an acceptor stem and a tRNA for aminoacylation. 总被引:3,自引:0,他引:3
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The anticodon-independent aminoacylation of RNA hairpin helices that reconstruct tRNA acceptor stems has been demonstrated for at least 10 aminoacyl-tRNA synthetases. For Escherichia coli cysteine tRNA synthetase, the specificity of aminoacylation of the acceptor stem is determined by the U73 nucleotide adjacent to the amino acid attachment site. Because U73 is present in all known cysteine tRNAs, we investigated the ability of the E. coli cystein enzyme to aminoacylate a heterologous acceptor stem. We show here that a minihelixCys based on the acceptor-T psi C stem of yeast tRNACys is a substrate for the E. coli enzyme, and that aminoacylation of this minihelix is dependent on U73. Additionally, we identify two base pairs in the acceptor stem that quantitatively convert the E. coli acceptor stem to the yeast acceptor stem. The influence of U73 and these two base pairs is completely retained in the full-length tRNA. This suggests a conserved relationship between the acceptor stem alone and the acceptor stem in the context of a tRNA for aminoacylation with cysteine. However, the primary determinant in the species-specific aminoacylation of the E. coli and yeast cysteine tRNAs is a tertiary base pair at position 15:48 outside of the acceptor stem. Although E. coli tRNACys has an unusual G15:G48 tertiary base pair, yeast tRNACys has a more common G15:C48 that prevents efficient aminoacylation of yeast tRNACys by the E. coli enzyme.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
8.
André Eichert Alexander Ulrich Volker A. Erdmann 《Biochemical and biophysical research communications》2010,395(3):291-536
We solved the X-ray structures of two Escherichia coli tRNASer acceptor stem microhelices. As both tRNAs are aminoacylated by the same seryl-tRNA-synthetase, we performed a comparative structure analysis of both duplexes to investigate the helical conformation, the hydration patterns and magnesium binding sites. It is well accepted, that the hydration of RNA plays an important role in RNA-protein interactions and that the extensive solvent content of the minor groove has a special function in RNA. The detailed comparison of both tRNASer microhelices provides insights into the structural arrangement of the isoacceptor tRNA aminoacyl stems with respect to the surrounding water molecules and may eventually help us to understand their biological function at atomic resolution. 相似文献
9.
Single sequence of a helix-loop peptide confers functional anticodon recognition on two tRNA synthetases. 总被引:1,自引:0,他引:1
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The specific aminoacylation of RNA oligonucleotides whose sequences are based on the acceptor stems of tRNAs can be viewed as an operational RNA code for amino acids that may be related to the development of the genetic code. Many synthetases also have direct interactions with tRNA anticodon triplets and, in some cases, these interactions are thought to be essential for aminoacylation specificity. In these instances, an unresolved question is whether interactions with parts of the tRNA outside of the anticodon are sufficient for decoding genetic information. Escherichia coli isoleucyl- and methionyl-tRNA synthetases are closely related enzymes that interact with their respective anticodons. We used binary combinatorial mutagenesis of a 10 amino acid anticodon binding peptide in these two enzymes to identify composite sequences that would confer function to both enzymes despite their recognizing different anticodons. A single peptide was found that confers function to both enzymes in vivo and in vitro. Thus, even in enzymes where anticodon interactions are normally important for distinguishing one tRNA from another, these interactions can be 'neutralized' without losing specificity of amino-acylation. We suggest that acceptor helix interactions may play a role in providing the needed specificity. 相似文献
10.
Inhibition of transfer messenger RNA aminoacylation and trans-translation by aminoglycoside antibiotics 总被引:2,自引:0,他引:2
Transfer messenger RNA (tmRNA) directs the modification of proteins of which the biosynthesis has stalled or has been interrupted. Here, we report that aminoglycosides can interfere with this quality control system in bacteria, termed trans-translation. Neomycin B is the strongest inhibitor of tmRNA aminoacylation with alanine (K(i) value of approximately 35 micro m), an essential step during trans-translation. The binding sites of neomycin B do not overlap with the identity determinants for alanylation, but the aminoglycoside perturbs the conformation of the acceptor stem that contains the aminoacylation signals. Aminoglycosides reduce the conformational freedom of the transfer RNA-like domain of tmRNA. Additional contacts between aminoglycosides and tmRNA are within the tag reading frame, probably also disturbing reprogramming of the stalled ribosomes prior protein tagging. Aminoglycosides impair tmRNA aminoacylation in the presence of all of the transfer RNAs from Escherichia coli, small protein B, and elongation factor Tu, but when both proteins are present, the inhibition constant is 1 order of magnitude higher. SmpB and elongation factor Tu have RNA chaperone activities, ensuring that tmRNA adopts an optimal conformation during aminoacylation. 相似文献
11.
Structural studies on tRNA acceptor stem microhelices: exchange of the discriminator base A73 for G in human tRNALeu switches the acceptor specificity from leucine to serine possibly by decreasing the stability of the terminal G1-C72 base pair.
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A U Metzger M Heckl D Willbold K Breitschopf U L RajBhandary P Rsch H J Gross 《Nucleic acids research》1997,25(22):4551-4556
Correct recognition of transfer RNAs (tRNAs) by aminoacyl-tRNA synthetases (aaRS) is crucial to the maintenance of translational fidelity. The discriminator base A73 in human tRNALeuis critical for its specific recognition by the aaRS. Exchanging A73 for G abolishes leucine acceptance and converts it into a serine acceptor in vitro . Two RNA microhelices of 24 nt length that correspond to the tRNALeuacceptor stem and differ only in the discriminator base were synthesized: a wild-type tRNALeumicrohelix, where nt 21 corresponds to the discriminator base position 73, and an A21G mutant microhelix. To investigate whether different identities of both tRNAs are caused by conformational differences, NMR and UV melting experiments were performed on both microhelices. Two-dimentional NOESY spectra showed both microhelices to exhibit the same overall conformation at their 3'-CCA ends. Thermodynamic analysis and melting behaviour of the base-paired imino protons observed by NMR spectroscopy suggest that the A21G (A73G in tRNA) exchange results in a decrease of melting transition cooperativity and a destabilization of the terminal G1-C20 (G1-C72 in tRNA) base pair. Furthermore, the fact that this 3'-terminal imino proton is more solvent-exposed at physiological temperature might be another indication for the importance of the stability of the terminal base pair for specific tRNA recognition. 相似文献
12.
13.
A biologically active 53 kDa fragment of overproduced alanyl-tRNA synthetase from Thermus thermophilus HB8 specifically interacts with tRNA Ala acceptor helix.
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The alaS gene encoding the alanyl-tRNA synthetase (AlaRS) from Thermus thermophilus HB8 was cloned and sequenced. The gene comprises 2646 bp, corresponding to 882 amino acids, 45% of which are identical to the enzyme from Escherichia coli . The T. thermophilus AlaRS was overproduced in E.coli , purified and characterized. It has high thermal stability up to approximately 65 degrees C, with a temperature optimum of aminoacylation activity at approximately 60 degrees C, and will be valuable for crystallization. The purified enzyme appears as a dimer with a specific activity of 220 U/mg and k cat/ K M values of 118 000/s/M for alanine and 114 000/s/M for ATP. By genetic engineering a 53 kDa fragment of AlaRS comprising the N-terminal 470 amino acids (AlaN470) was also overproduced and purified. It is as stable as entire AlaRS and sufficient for specific aminoacylation of intact tRNAAla, as well as acceptor stem microhelices with a G3-U70, but not U3-A70, I3-U70 or C3-U70, base pair. The reduced binding strength of such microhelices to AlaN470 enabled, due to the resulting fast exchange of the microhelices between free and complexed states, preliminary NMR analyses of the binding mode and intermolecular recognition. 相似文献
14.
Mutant aminoacyl-tRNA synthetase that compensates for a mutation in the major identity determinant of its tRNA 总被引:1,自引:0,他引:1
A single G3.U70 base pair in the acceptor helix is the major determinant for the identity of alanine transfer RNAs (Hou & Schimmel, 1988). Introduction of this base pair into foreign tRNA sequences confers alanine acceptance on them. Moreover, small RNA helices with as few as seven base pairs can be aminoacylated with alanine, provided that they encode the critical base pair (Francklyn & Schimmel, 1989). Alteration of G3.U70 to G3.C70 abolishes aminoacylation with alanine in vivo and in vitro. We describe here the mutagenesis and selection of a single point mutation in Escherichia coli Ala-tRNA synthetase that compensates for a G3.C70 mutation in tRNAAla. The mutation maps to a region previously implicated as proximal to the acceptor end of the bound tRNA. In contrast to the wild-type enzyme, the mutant charges small RNA helices that encode a G3.C70 base pair. However, the mutant enzyme retains specificity for alanine tRNA and can serve as the sole source of Ala-tRNA synthetase in vivo. The results demonstrate the capacity of an aminoacyl-tRNA synthetase to compensate through a single amino acid substitution for mutations in the major determinant of its cognate tRNA. 相似文献
15.
Role of acceptor stem conformation in tRNAVal recognition by its cognate synthetase. 总被引:1,自引:0,他引:1
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Although the anticodon is the primary element in Escherichia coli tRNAValfor recognition by valyl-tRNA synthetase (ValRS), nucleotides in the acceptor stem and other parts of the tRNA modulate recognition. Study of the steady state aminoacylation kinetics of acceptor stem mutants of E.coli tRNAValdemonstrates that replacing any base pair in the acceptor helix with another Watson-Crick base pair has little effect on aminoacylation efficiency. The absence of essential recognition nucleotides in the acceptor helix was confirmed by converting E.coli tRNAAlaand yeast tRNAPhe, whose acceptor stem sequences differ significantly from that of tRNAVal, to efficient valine acceptors. This transformation requires, in addition to a valine anticodon, replacement of the G:U base pair in the acceptor stem of these tRNAs. Mutational analysis of tRNAValverifies that G:U base pairs in the acceptor helix act as negative determinants of synthetase recognition. Insertion of G:U in place of the conserved U4:A69 in tRNAValreduces the efficiency of aminoacylation, due largely to an increase in K m. A smaller but significant decline in aminoacylation efficiency occurs when G:U is located at position 3:70; lesser effects are observed for G:U at other positions in the acceptor helix. The negative effects of G:U base pairs are strongly correlated with changes in helix structure in the vicinity of position 4:69 as monitored by19F NMR spectroscopy of 5-fluorouracil-substituted tRNAVal. This suggests that maintaining regular A-type RNA helix geometry in the acceptor stem is important for proper recognition of tRNAValby valyl-tRNA synthetase.19F NMR also shows that formation of the tRNAVal-valyl-tRNA synthetase complex does not disrupt the first base pair in the acceptor stem, a result different from that reported for the tRNAGln-glutaminyl-tRNA synthetase complex. 相似文献
16.
Conformational requirements of tobacco mosaic virus RNA for aminoacylation and adenylation. 总被引:2,自引:0,他引:2
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The RNA conformational requirements for both aminoacylation and adenylation emerging from our studies performed using the valine- and the tyrosine-accepting plant viral RNAs are now strongly supported by the histidine-accepting tobacco mosaic virus RNA: an 'L'-shaped conformation is recognized by the aminoacyl-tRNA synthetase whereas only the aminoacyl RNA domain (equivalent in tRNAs to the continuous helix formed by the acceptor stem and the T stem and loop) interacts with the tRNA nucleotidyltransferase. 相似文献
17.
The relationship of thermodynamic stability at a G x U recognition site to tRNA aminoacylation specificity
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The G x U pair at the third position in the acceptor helix of Escherichia coli tRNA(Ala) is critical for aminoacylation. The features that allow G x U recognition are likely to include direct interaction of alanyl-tRNA synthetase with distinctive atomic groups and indirect recognition of the structural and stability information encoded in the sequence of G x U and its immediate context. The present work investigates the thermodynamic stability and acceptor activity for a comprehensive set of variant RNAs with substitutions of the G x U pair of E. coli tRNA(Ala). The four RNAs with Watson-Crick substitutions had a lower acceptor activity and a higher stability relative to the G x U RNA. On the other hand, the RNAs with mispair substitutions had a lower stability, but either a higher or a lower acceptor activity. Thus, the entire set of variant RNAs does not exhibit a correlation between thermodynamic stability of the free, unbound tRNA and its acceptor activity. The substantial acceptor activity of tRNAs with particular mispair substitutions may be explained by their ability to assume the conformational preferences of alanyl-tRNA synthetase. Moreover, the G x U pair may provide a point of deformability for the substrate tRNA to adapt to the enzyme's active site. 相似文献
18.
Identification of discriminator base atomic groups that modulate the alanine aminoacylation reaction
Fischer AE Beuning PJ Musier-Forsyth K 《The Journal of biological chemistry》1999,274(52):37093-37096
Specific aminoacylation of tRNAs involves activation of an amino acid with ATP followed by amino acid transfer to the tRNA. Previous work showed that the transfer of alanine from Escherichia coli alanyl-tRNA synthetase to a cognate RNA minihelix involves a transition state sensitive to changes in the tRNA acceptor stem. Specifically, the "discriminator" base at position 73 of minihelix(Ala) is a critical determinant of the transfer step of aminoacylation. This single-stranded nucleotide has previously been shown by solution NMR to be stacked predominantly onto G(1) of the first base pair of the alanine acceptor stem helix. In this work, RNA duplex(Ala) variants were prepared to investigate the role of specific discriminator base atomic groups in aminoacylation catalytic efficiency. Results indicate that the purine structure appears to be important for stabilization of the transition state and that major groove elements are more critical than those located in the minor groove. This result is in accordance with the predicted orientation of a class II synthetase at the end of the acceptor helix. In particular, substitution of the exocyclic amino group of A(73) with a keto-oxygen resulted in negative discrimination at this site. Taken together, these new results are consistent with the involvement of major groove atomic groups of the discriminator base in the formation of the transition state for the amino acid transfer step. 相似文献
19.
20.
Transfer RNAs for primordial amino acids contain remnants of a primitive code at position 3 to 5 总被引:2,自引:0,他引:2
Analysis of the nucleotide sequence of 1,400 transfer RNAs has revealed the imprint of a prototypic genetic code in position 3-4-5 of the acceptor stem. It appears only in the transfer RNAs for the primordial amino acids ie those found by chemical condensation of a nitrogen-methane-water-ammonia mixture. The model for primitive protein synthesis as mentioned by Crick assumes a direct interaction between the amino acid and a prototypic adaptor oligonucleotide. This has hitherto appeared irreconcilable with the large spatial separation between the aminoacylation site and the anticodon in present day transfer RNAs. The observations reported here show how this paradox can be resolved by a process of duplication and cleavage of a prototypic adaptor. 相似文献