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Using in vitro tRNA transcripts and minihelices it was shown that the tyrosine identity for tRNA charging by tyrosyl-tRNA synthetase (TyrRS) from the archaeon Methanococcus jannaschii is determined by six nucleotides: the discriminator base A73 and the first base-pair C1-G72 in the acceptor stem together with the anticodon triplet. The anticodon residues however, participate only weakly in identity determination, especially residues 35 and 36. The completeness of the aforementioned identity set was verified by its tranfer into several tRNAs which then become as efficiently tyrosylatable as the wild-type transcript from M. jannaschii. Temperature dependence experiments on both the structure and the tyrosylation properties of M. jannaschii and yeast tRNA(Tyr) transcripts show that the archaeal transcript has greater structural stability and enhanced aminoacylation behaviour than the yeast transcript. Tyrosine identity in M. jannaschii is compared to that in yeast, and the conservation of the major determinant in both organisms, namely the C1-G72 pair, gives additional support to the existence of a functional connection between archaeal and eukaryotic aminoacylation systems.
Nucleoside modifications are important to the structure of all tRNAs and are critical to the function of some tRNA species. The transcript of human tRNA(Lys3)(UUU) with a UUU anticodon, and the corresponding anticodon stem and loop domain (ASL(Lys3)(UUU)), are unable to bind to poly-A programmed ribosomes. To determine if specific anticodon domain modified nucleosides of tRNA(Lys) species would restore ribosomal binding and also affect thermal stability, we chemically synthesized ASL(Lys) heptadecamers and site-specifically incorporated the anticodon domain modified nucleosides pseudouridine (Psi(39)), 5-methylaminomethyluridine (mnm(5)U(34)) and N6-threonylcarbamoyl-adenosine (t(6)A(37)). Incorporation of t(6)A(37) and mnm(5)U(34) contributed structure to the anticodon loop, apparent by increases in DeltaS, and significantly enhanced the ability of ASL(Lys3)(UUU) to bind poly-A programmed ribosomes. Neither ASL(Lys3)(UUU)-t(6)A(37) nor ASL(Lys3)(UUU)-mnm(5)U(34) bound AAG programmed ribosomes. Only the presence of both t(6)A(37) and mnm(5)U(34) enabled ASL(Lys3)(UUU) to bind AAG programmed ribosomes, as well as increased its affinity for poly-A programmed ribosomes to the level of native Escherichia coli tRNA(Lys). The completely unmodified anticodon stem and loop of human tRNA(Lys1,2)(CUU) with a wobble position-34 C bound AAG, but did not wobble to AAA, even when the ASL was modified with t(6)A(37). The data suggest that tRNA(Lys)(UUU) species require anticodon domain modifications in the loop to impart an ordered structure to the anticodon for ribosomal binding to AAA and require a combination of modified nucleosides to bind AAG.
The interaction between fMet-tRNA(f)(Met) and Bacillus stearothermophilus translation initiation factor IF2 has been characterized. We demonstrate that essentially all thermodynamic determinants governing the stability and the specificity of this interaction are localized within the acceptor hexanucleotide fMet-3'ACCAAC of the initiator tRNA and a fairly small area at the surface of the beta-barrel structure of the 90-amino acid C-terminal domain of IF2 (IF2 C-2). A weak but specific interaction between IF2 C-2 and formyl-methionyl was also demonstrated. The surface of IF2 C-2 interacting with fMet-tRNA(f)(Met) has been mapped using two independent approaches, site- directed mutagenesis and NMR spectroscopy, which yielded consistent results. The binding site comprises C668 and G715 located in a groove accommodating the methionyl side-chain, R700, in the vicinity of the formyl group, Y701 and K702 close to the acyl bond between fMet and tRNA(f)(Met), and the surface lined with residues K702-S660, along which the acceptor arm of the initiator tRNA spans in the direction 3' to 5'.
Naturally occurring nucleoside modifications are an intrinsic feature of transfer RNA (tRNA), and have been implicated in the efficiency, as well as accuracy-of codon recognition. The structural and functional contributions of the modified nucleosides in the yeast tRNA(Phe) anticodon domain were examined. Modified nucleosides were site-selectively incorporated, individually and in combinations, into the heptadecamer anticodon stem and loop domain, (ASL(Phe)). The stem modification, 5-methylcytidine, improved RNA thermal stability, but had a deleterious effect on ribosomal binding. In contrast, the loop modification, 1-methylguanosine, enhanced ribosome binding, but dramatically decreased thermal stability. With multiple modifications present, the global ASL stability was mostly the result of the individual contributions to the stem plus that to the loop. The effect of modification on ribosomal binding was not predictable from thermodynamic contributions or location in the stem or loop. With 4/5 modifications in the ASL, ribosomal binding was comparable to that of the unmodified ASL. Therefore, modifications of the yeast tRNA(Phe) anticodon domain may have more to do with accuracy of codon reading than with affinity of this tRNA for the ribosomal P-site. In addition, we have used the approach of site-selective incorporation of specific nucleoside modifications to identify 2'O-methylation of guanosine at wobble position 34 (Gm34) as being responsible for the characteristically enhanced chemical reactivity of C1400 in Escherichia coli 16S rRNA upon ribosomal footprinting of yeast tRNA(Phe). Thus, effective ribosome binding of tRNA(Phe) is a combination of anticodon stem stability and the correct architecture and dynamics of the anticodon loop. Correct tRNA binding to the ribosomal P-site probably includes interaction of Gm34 with 16S rRNA C1400.
An improved method for the rapid separation of aminoacyl-tRNA from tRNA by chromatography on dihydroxyboryl-substituted cellulose has been developed. The method relies on the selective binding of unacylated tRNA to the cell cellulose support containing dihydroxyboryl groups. This binding is the result of complex formation between the cis-diol group of the 3'-terminal ribose in tRNA and the dihydroxyboryl groups immobilized on the resin. Aminoacyl-tRNA cannot undergo borate complex formation and is not retained on the resin. The separation is carried out at near neutral pH values ensuring stability of the aminoacyl ester linkage. The aminoacyl-tRNAs are obtained in very high purity. Aminoacyl-tRNA species containing the modified nucleoside Q are also retained on dihydroxyboryl cellulose. Conditions for isolating all Q base containing tRNA species from unfractionated tRNA are described.
Genomic size of animal mitochondrial DNA is usually minimized over time. Thus, when regional duplications occur, they are followed by a rapid elimination of redundant material. In contrast to this general view, we report here long-sustained tRNA pseudogenes in the mitochondrial genome (mitogenome) of teleost fishes of the family Scaridae (parrotfishes). During the course of a molecular phylogenetic study of the suborder Labroidei, we determined the complete nucleotide sequence of the mitogenome for a parrotfish, Chlorurus sordidus, and found a gene rearrangement accompanied by a tRNA pseudogene. In the typical gene order of vertebrates, a tRNA-gene cluster between ND1 and ND2 genes includes tRNA(Ile) (I), tRNA(Gln) (Q), and tRNA(Met) (M) genes in this order (IQM). However, in the mitogenome of the parrotfish, the tRNA(Met) gene was inserted between the tRNA(Ile) and the tRNA(Gln) genes, and the tRNA(Gln) gene was followed by a putative tRNA(Met) pseudogene (psiM). Such a tRNA gene rearrangement including a pseudogene (IMQpsiM) was found in all of the 10 examined species, representing 7 of the 10 currently recognized scarid genera. All sister groups examined (20 species of Labridae and a single species of Odacidae) had the typical gene order of vertebrate mitogenomes. Phylogenetic analysis of the tRNA(Met) genes and the resulting pseudogenes demonstrated that the ancestral tRNA(Met) gene was duplicated in a common ancestor of the parrotfish. Based on the fossil record, these results indicate that the pseudogenes have survived at least 14 million years. Most of the vertebrate mitochondrial gene rearrangements involving the IQM region have held the tRNA(Met) gene just upstream of the ND2 gene, and even in a few exceptional cases, including the present ones, the tRNA pseudogenes have been found in that position. In addition, most of these tRNA(Met) pseudogenes maintained clover-leaf secondary structures, with the remainder sustaining the clover-leaf structure in the "top half (TpsiC and acceptor arms). Considering their potential secondary structures (holding "top halves" of the clover-leaf structures), locations within mitogenomes (flanking the 5' ends of the ND2 genes) and stabilities over time (survived at least 14 Myr), it is likely that the tRNA pseudogenes retain function as punctuation marks for mitochondrial ND2 mRNA processing.
Overproducing Escherichia coli tRNAGlu in its homologous host results in the presence of several distinctly modified forms of this molecule that we name modivariants. The predominant tRNAGlu modivariant in wild-type E. coli contains five modified nucleosides: Psi13, mnm5s2U34, m2A37, T54 and Psi55. Four other overproduced modivariants differ from it by, respectively, either the presence of an additional Psi, or the presence of s2U34, or the lack of A37 methylation combined with either s2U34 or U34. Chemical probing reveals that the anticodon loop of the predominant modivariant is less reactive to the probes than that of the four others. Furthermore, the modivariant with neither mnm5s2U34 nor m2A37 has additional perturbations in the D- and T-arms and in the variable region. The lack of a 2-thio group in nucleoside 34, which is mnm5s2U in the predominant tRNAGlu modivariant, decreases by 520-fold the specificity of E. coli glutamyl-tRNA synthetase for tRNAGlu in the aminoacylation reaction, showing that this thio group is the identity element in the modified wobble nucleotide of E. coli tRNAGlu. The modified nucleosides content also influences the recognition of ATP and glutamate by this enzyme, and in this case also, the predominant modivariant is the one that allows the best specificity for these two substrates. These structural and kinetic properties of tRNAGlu modivariants indicate that the modification system of tRNAGlu optimizes the stability of tRNAGlu and its action as cofactor of the glutamyl-tRNA synthetase for the recognition of glutamate and ATP.
The Neurospora CYT-18 protein, a tyrosyl-tRNA synthetase, which functions in splicing group I introns in mitochondria, promotes splicing of mutants of the distantly related bacteriophage T4 td intron. In an in vivo assay, wild-type CYT-18 protein expressed in E. coli suppressed mutations in the td intron's catalytic core. CYT-18-suppressible mutations were also suppressed by high Mg2+ or spermidine in vitro, suggesting they affect intron structure. Both the N- and C-terminal domains of CYT-18 are required for efficient splicing, but CYT-18 with a large C-terminal truncation retains some activity. Our results indicate that CYT-18 interacts with conserved structural features of group I introns, and they provide direct evidence that a protein promotes splicing by stabilizing the catalytically active structure of the intron RNA.
Escherichia coli methionyl-tRNA synthetase consists of catalytic, anticodon-binding, and dimerization domains. The polypeptide was genetically cleaved and expressed as multiple subunits to investigate how peptide breakage affects the activity and stability of the enzyme. Mutants cleaved near conserved or functionally important sites were inactive. A few bipartite mutants were active, but they showed temperature sensitivity in their activity and stability. An additional cleavage of the active bipartites inactivated the enzyme, suggesting that at least two functional domains have to be covalently connected to retain the activity. This approach proves to be useful in determining the structural and functional organization of a protein.
Human immunodeficiency virus (HIV-1) exclusively selects tRNA(Lys,3) as the primer for initiation of reverse transcription. How and why HIV-1 selects the tRNA is unresolved. To address this issue, we have generated HIV-1 in which the PBS was changed to be complementary to alternative tRNAs. In this study, we report on HIV-1 that have the PBS mutated to be complementary to tRNA(Thr), tRNA(Phe), tRNA(Ser) and tRNA(Tyr). Virus with a PBS complementary to tRNA(Thr) grew slightly slower than the wild type virus and maintained the PBS for an extended culture period before finally reverting back to utilize tRNA(Lys,3). In contrast, viruses with a PBS complementary to tRNA(Phe) or tRNA(Ser) rapidly reverted to utilize tRNA(Lys,3) following limited in vitro replication, while a virus with a PBS complementary to tRNA(Tyr) had severely compromised infectivity and did not productively infect a continuous T cell line (SupT1) or human peripheral blood mononuclear cells (PBMC). Modification of the A-loop region to be complementary to tRNA(Thr) with the mutation in the PBS to be complementary to tRNA(Thr) resulted in a virus that could stably utilize this tRNA while the modification of the A-loop to be complementary to the anticodon of tRNA(Ser) did not allow the virus to stably utilize tRNA(Ser). Modification of the A-loop region to be complementary to the anticodon of tRNA(Phe) severely impacted the replication of this virus. Finally, the modification of the A-loop region to be complementary to tRNA(Tyr) did not rescue the virus with a PBS complementary to tRNA(Tyr). The results of these studies demonstrate the diverse effects that alteration of the PBS to force selection of alternative primers have on HIV-1 replication and provide a framework to understand the dynamics of primer selection.
We showed previously that the tRNA tertiary structure makes an important contribution to the identity of yeast tRNA(Met) (Senger B, Aphasizhev R, Walter P, Fasiolo F, 1995, J Mol Biol 249:45-58). To learn more about the role played by the tRNA framework, we analyzed the effect of some phosphodiester cleavages and 2'OH groups in tRNA binding and aminoacylation. The tRNA is inactivated provided the break occurs in the central core region responsible for the tertiary fold or in the anticodon stem/loop region. We also show that, for tRNA(Met) to bind, the anticodon loop, but not the anticodon stem, requires a ribosephosphate backbone. A tertiary mutant of yeast tRNA(Met) involving interactions from the D- and T-loop unique to the initiator species fails to be aminoacylated, but still binds to yeast methionyl-tRNA synthetase. In the presence of 10 mM MgCl2, the mutant transcript has a 3D fold significantly stabilized by about 30 degrees C over a wild-type transcript as deduced from the measure of their T(m) values. The k(cat) defect of the tRNA(Met) mutant may arise from a failure to overcome an increase of the free energetic cost of distorting the more stable tRNA structure and/or a tRNA based MetRS conformational change required for formation of transition state of aminoacylation.
The T box and S box transcription termination control systems are widely used for control of gene expression in Gram-positive bacteria, but are rare in Gram-negative organisms. Both of these systems can be recognized in genomic data because of high conservation of primary sequence and structural elements. The T box system regulates a variety of amino acid-related genes, while the S box system is dedicated to genes involved in methionine metabolism. While both systems involve gene regulation at the level of premature termination of transcription, the molecular mechanisms employed are very different. In the T box system, expression is induced by stabilization of an antiterminator structure in the leader by interaction with the cognate uncharged tRNA; this prevents formation of the competing terminator helix, allowing synthesis of the full-length mRNA. Disruption of conserved leader features results in loss of readthrough. In the S box system, the antiterminator form of the leader is the more stable form. A competing anti-antiterminator must be stabilized by an unknown factor during growth in methionine to prevent formation of the antiterminator, thereby allowing formation of the terminator helix. Disruption of conserved leader elements results in constitutive expression.
tRNA halves are among the most abundant short non-coding RNAs in the cellular transcriptome. Here we report that in androgen receptor-positive LNCaP prostate cancer cells, the hormone-dependent 5'-tRNALysCUU half promoted cell proliferation by facilitating cell cycle progression. Global mRNA profiling upon the 5'-tRNALysCUU half depletion revealed that the mRNA of p21, a negative regulator of the cell cycle, is post-transcriptionally destabilized via a 5'-tRNALysCUU half-driven mechanism. YBX1, identified as a protein interacting with 5'-tRNALysCUU half in the cytosol, was shown to stabilize p21 mRNA. Specific sequences resembling the 5'-tRNALysCUU half, located in the 3'-UTR of p21 mRNA and termed LL588, were identified as the binding site for YBX1 and are required for p21 mRNA stability. In vitro binding assays demonstrated that the 5'-tRNALysCUU half is capable of displacing YBX1 from LL588. Collectively, our findings suggest that the 5'-tRNALysCUU half directly binds to and displaces YBX1 from p21 mRNA, leading to the destabilization of p21 mRNA and the promotion of cell cycle progression in hormone-dependent cancers. Our study illuminates the role of tRNA halves in regulating mRNA stability and suggests that this may be part of broader regulatory networks affecting mRNA levels, orchestrated by various tRNA halves and their interacting proteins.
To investigate the role of alpha helices in protein thermostability, we compared energy characteristics of alpha helices from thermophilic and mesophilic proteins belonging to four protein families of known three-dimensional structure, for at least one member of each family. The changes in intrinsic free energy of alpha-helix formation were estimated using the statistical mechanical theory for describing helix/coil transitions in peptide helices [Munoz, V., Serrano, L. Nature Struc. Biol. 1:399-409, 1994; Munoz, V., Serrano, L. J. Mol. Biol. 245:275-296, 1995; Munoz, V., Serrano, L. J. Mol. Biol. 245:297-308, 1995]. Based on known sequences of mesophilic and thermophilic RecA proteins we found that (1) a high stability of alpha helices is necessary but is not a sufficient condition for thermostability of RecA proteins, (2) the total helix stability, rather than that of individual helices, is the factor determining protein thermostability, and (3) two facets of intrahelical interactions, the intrinsic helical propensities of amino acids and the side chain-side chain interactions, are the main contributors to protein thermostability. Similar analysis applied to families of L-lactate dehydrogenases, seryl-tRNA synthetases, and aspartate amino transferases led us to conclude that an enhanced total stability of alpha helices is a general feature of many thermophilic proteins. The magnitude of the observed decrease in intrinsic free energy on alpha-helix formation of several thermoresistant proteins was found to be sufficient to explain the experimentally determined increase of their thermostability. Free energies of intrahelical interactions of different RecA proteins calculated at three temperatures that are thought to be close to its normal environmental conditions were found to be approximately equal. This indicates that certain flexibility of RecA protein structure is an essential factor for protein function. All RecA proteins analyzed fell into three temperature-dependent classes of similar alpha-helix stability (delta G(int) = 45.0 +/- 2.0 kcal/mol). These classes were consistent with the natural origin of the proteins. Based on the sequences of protein alpha helices with optimized arrangement of stabilizing interactions, a natural reserve of RecA protein thermoresistance was estimated to be sufficient for conformational stability of the protein at nearly 200 degrees C.
The ribonuclease resistance assay has been used to probe the effect of trypsin modification of the Escherichia coli elongation factor Tu X GTP on the interaction with E. coli aminoacyl-tRNAs. First, the equilibrium dissociation constant of the trypsin-modified Tu X GTP X Thr-tRNA complex was determined to be 2.3 (0.1) X 10(-5)M at 4 degrees C, pH 7.4. Second, binding of 17 of 20 noninitiator aminoacyl-tRNAs and four sets of purified isoacceptor tRNAs to the modified protein was measured. At 4 degrees C, the complex stabilities vary 500-fold over the range of aminoacyl-tRNAs, with Gln-tRNA forming the strongest ternary complex and Val-tRNA, the weakest. The results are compared to a similar study of ternary complex formation using intact elongation factor Tu X GTP, and the major differences are discussed. An analysis of both data sets, particularly that for the leucine isoacceptor tRNAs, suggests that the trypsin modification of elongation factor Tu X GTP disrupts a region of protein that is involved with the aminoacyl side chain rather than that of the acceptor stem helix region of the aminoacyl-tRNA.
In a hyperthermophilic bacterium, Aquifex aeolicus, leucyl-tRNA synthetase (LeuRS) consists of two non-identical polypeptide subunits (alpha and beta), different from the canonical LeuRS, which has a single polypeptide chain. By PCR, using genome DNA of A. aeolicus as a template, genes encoding the alpha and beta subunits were amplified and cloned in Escherichia coli. The alpha subunit could not be expressed stably in vivo, whereas the beta subunit was overproduced and purified by a simple procedure. The beta subunit was inactive in catalysis but was able to bind tRNA(Leu). Interestingly, the heterodimer alphabeta-LeuRS could be overproduced in E. coli cells containing both genes and was purified to 95% homogeneity as a hybrid dimer. The kinetics of A. aeolicus LeuRS in pre-steady and steady states and cross-recognition of LeuRS and tRNA(Leu) from A. aeolicus and E. coli were studied. Magnesium concentration, pH value, and temperature aminoacylation optima were determined to be 12 mm, 7.8, and 70 degrees C, respectively. Under optimal conditions, A. aeolicus alphabeta-LeuRS is stable up to 65 degrees C.
Although aminoacyl-tRNA synthetases (ARSs) are housekeeping enzymes essential for protein synthesis, they can play non-catalytic roles in diverse biological processes. Some ARSs are capable of forming complexes with each other and additional proteins. This characteristic is most pronounced in mammals, which produce a macromolecular complex comprising nine different ARSs and three additional factors: p43, p38 and p18. We have been aware of the existence of this complex for a long time, but its structure and function have not been well understood. The only apparent distinction between the complex-forming ARSs and those that do not form complexes is their ability to interact with the three non-enzymatic factors. These factors are required not only for the catalytic activity and stability of the associated ARSs, such as isoleucyl-, methionyl-, and arginyl-tRNA synthetase, but also for diverse signal transduction pathways. They may thus have joined the ARS community to coordinate protein synthesis with other biological processes.