Properties of tyrosyl transfer ribonucleic acid synthetase from two tyrS mutants of Escherichia coli K-12.
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Amino Acyl-tRNA Synthetases↗
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Determination of the amino acid sequence of rabbit, human, and wheat germ protein synthesis factor eIF-4C by cloning and chemical sequencing.
The small eukaryotic initiation factor (eIF)-4C is implicated in the initiation pathway, where it enhances ribosome dissociation into subunits and stabilizes the binding of the initiator Met-tRNA(i) to 40 S ribosomal subunits. In order to elucidate the function of eIF-4C, its structure has been further characterized. The amino acid sequence of many peptides from rabbit reticulocyte and wheat germ eIF-4C have been determined chemically. From the chemical sequencing of the rabbit protein, it was noted that at least two different eIF-4C molecules were present which differed by conservative substitutions at three positions (2 aspartic acid for glutamic acid switches and 1 valine for isoleucine switch). By the use of unique sequences with low codon degeneracy, primers were used to obtain a polymerase chain reaction product of appropriate size and sequence. This product was then used to isolate full-length coding sequence cDNA clones for human eIF-4C. A similar strategy was used to design PCR primers and then isolate a wheat cDNA clone which lacked the coding region for the first 23 amino acids, but contained a complete 3'-untranslated region. The protein amino acid sequence of wheat germ eIF-4C is 68% identical with the mammalian protein, and, allowing for the most conservative substitutions, the proteins are 76% similar. Both the mammalian and wheat germ proteins are 143 amino acids in length and have molecular weights of about 16,400. A unique feature of eIF-4C is its apparent "polarity" as 9 of the first 15 amino acids are basic while 13 of the last 20 amino acids are acidic. This dipole nature may enable the protein to interact with both the ribosome (perhaps via the rRNA) and other translation initiation factors.
Amino Acid Sequence↗
The thermodynamic stability of RNA duplexes and hairpins containing N6-alkyladenosines and 2-methylthio-N6-alkyladenosines.
The N6-alkyladenosines and 2-methylthio-N6-alkyladenosines make up over half of the population of all naturally modified adenosines and they are present in the transfer ribonucleic acids (tRNA) at position 37. We measured effects of N6-alkyladenosines and 2-methylthio-N6-alkyladenosines on the thermodynamic stability of RNA duplexes containing a U-A(Mod) base pair at internal and terminal duplex positions, as well as containing modified adenosines as a 3'-terminal unpaired nucleotide. Beside naturally modified adenosines such as N6-isopentenyladenosine (i6A), N6-methyladenosine (m6A), 2-methylthio-N6-isopentenyladenosine (ms2i6A) and 2-methylthio-N6-methyladenosine (ms2m6A), we studied several artificial modifications to evaluate the steric and electronic effects of N6-alkyl substituents. Moreover, some N6-alkyladenosines and 2-methylthio-N6-alkyladenosines were placed in hairpins at positions corresponding to nucleotide 37 of the tRNA anticodon arm, and the thermodynamic stability of those hairpins was studied. The stability of the modified RNA hairpins was measured in standard melting buffer containing 1 M sodium chloride as well as in physiological buffer containing 10 mM magnesium chloride and 150 mM potassium chloride. The results obtained indicate that the nature of the adenosine modification and the position of U-A(Mod) base pairs within the duplex influence the thermodynamic stability of RNA duplexes. For most of the modification, the destabilization of duplexes was observed. Moreover, we found that the buffer composition and the structure of the modified adenosine very significantly affect the thermodynamic stability of RNA.
Adenosine↗
The in vivo stability, maturation and aminoacylation of anticodon-substituted Escherichia coli initiator methionine tRNAs.
We have constructed eight anticodon-modified Escherichia coli initiator methionine (fMet) tRNAs by insertion of synthetic ribotrinucleotides between two fragments ('half molecules') derived from the initiator tRNA. The trinucleotides, namely CAU (the normal anticodon), CAA, CAC, CAG, GAA, GAC, GAG and GAU, were joined to the 5' and 3' tRNA fragments with T4 RNA ligase. The strategy of reconstruction permitted the insertion of radioactive 32P label between nucleotides 36 and 37. tRNAs were microinjected into the cytoplasm of Xenopus laevis oocytes, and the following properties were evaluated: the stability of these eubacterial tRNA variants in the eukaryotic oocytes; the enzymatic modification of the adenosine at position 37 (3' adjacent to the anticodon) and aminoacylation of the chimeric tRNAs by endogenous oocyte aminoacyl-tRNA synthetases. In contrast to other variants, the two RNAs having CAU and GAU anticodons were stable and underwent quantitative modification at A-37. These results show that the enzyme responsible for the modification of A-37 to N-[N-(9-beta-D-ribofuranosylpurine-6-yl)carbamoyl]threonine (t6A) is present in the cytoplasm of oocytes and is very sensitive to the anticodon environment of the tRNA. Also, these same GAU and CAU anticodon-containing tRNAs are fully aminoacylated with the heterologous oocyte aminoacyl-tRNA synthetases in vivo. During the course of this work we developed a generally applicable assay for the aminoacylation of femtomole amounts of labelled tRNAs.
Animals↗
Mapping the stability determinants of bacterial tyrosyl transfer RNA synthetases by an experimental evolutionary approach.
The tyrosyl-tRNA synthetases from Bacillus stearothermophilus (Bst-TyrTS) and Escherichia coli (Eco-TyrTS) are 56% identical in amino acid sequence. To map and characterize the set of interactions that makes Bst-TyrTS more stable than Eco-TyrTS, a family of nine hybrid proteins was constructed between the two enzymes. The N-terminal part of each hybrid came from Eco-TyrTS and the C-terminal part from Bst-TyrTS. The stability and activity of these hybrids were estimated by experiments of thermal inactivation and tRNA charging. For all the hybrids, the temperature of half-inactivation in 30 minutes was above 44 degrees C and the rate of charging was at least 40% that of Bst-TyrTS. In general, the temperature of half-inactivation increased and the rate of charging decreased monotonically when the number of residues coming from the more stable and less active Bst-TyrTS increased. As a result, the rate of charging decreased when the temperature of half-inactivation increased. These results show that the sequences and structures of the two enzymes can replace each other locally and still give a stable and active TyrTS, and that the greater stability of Bst-TyrTS is due to cumulative changes of residues scattered along the sequence. They suggest that Bst-TyrTS is more rigid than Eco-TyrTS at low temperature. The existence of a few exceptional hybrids, having stabilities or activities lower than those of the neighbouring hybrids, shows that compensatory changes of residues have occurred between the two sequences during evolution. These exceptions could be explained by the systematic identification of the couples of residues that are in contact in the Bst-TyrTS structure and become heterologous in some hybrids.
Binding Sites↗
The glutamyl-tRNA synthetase of Escherichia coli contains one atom of zinc essential for its native conformation and its catalytic activity.
The glutamyl-tRNA synthetase of Escherichia coli contains one atom of zinc. This metal ion is strongly bound, as it is not removed by 8 M urea. Slow removal of the zinc at 4 degrees C in the presence of the specific chelating agent, 1,10-phenanthroline, is proportional to the loss of aminoacylation activity and to the presence of a more open conformer of the enzyme. This conformer migrates more slowly than the native enzyme during gel electrophoresis under nondenaturing conditions and binds tRNA(Glu). Infrared spectroscopy measurements show that it differs from the native enzyme by a lower alpha-helix content and a higher proportion of beta-sheet and unordered structures. ATP protects the enzyme against 1,10-phenanthroline-mediated zinc removal, suggesting that the zinc-binding region is closely associated with the catalytic site. Additional support for this conclusion comes from the presence of zinc in the 27-kDa N-terminal half of the enzyme and in a 10-kDa fragment. The latter is homologous to the tRNA acceptor helix binding domain of E. coli glutaminyl-tRNA synthetase. The presence of the conserved CYC motif in this domain of the zinc-containing glutamyl-tRNA synthetases of E. coli and Bacillus subtilis, and its absence in that of Thermus thermophilus and the E. coli glutaminyl-tRNA synthetase which do not contain zinc, suggest that the cysteines of this motif and the C- and H-rich 125CRHSHEHHX5C138 segment present in the 10-kDa zinc-binding fragment are involved in zinc binding by the glutamyl-tRNA synthetase of E. coli.
Amino Acid Sequence↗
A reduced level of charged tRNAArgmnm5UCU triggers the wild-type peptidyl-tRNA to frameshift.
Frameshift mutations can be suppressed by a variety of differently acting external suppressors. The +1 frameshift mutation hisC3072, which has an extra G in a run of Gs, is corrected by the external suppressor mutation sufF44. We have shown that sufF44 and five additional allelic suppressor mutations are located in the gene argU coding for the minor tRNAArgmnm5UCU and alter the secondary and/or tertiary structure of this tRNA. The C61U, G53A, and C32U mutations influence the stability, whereas the C56U, C61U, G53A, and G39A mutations decrease the arginylation of tRNAArgmnm5UCU. The T-10C mutant has a base substitution in the -10 consensus sequence of the argU promoter that reduces threefold the synthesis of tRNAArgmnm5UCU . The lower amount of tRNAArgmnm5UCU or impaired arginylation, either independently or in conjunction, results in inefficient reading of the cognate AGA codon that, in turn, induces frameshifts. According to the sequence of the peptide produced from the suppressed -GGG-GAA-AGA- frameshift site, the frameshifting tRNA in the argU mutants is tRNAGlumnm5s2UUC, which decodes the GAA codon located upstream of the AGA arginine codon, and not the mutated tRNAArgmnm5UCU. We propose that an inefficient decoding of the AGA codon by a defective tRNAArgmnm5UCU stalls the ribosome at the A-site codon allowing the wild-type form of peptidyl-tRNAGlumnm5s2UUC to slip forward 1 nucleotide and thereby re-establish the ribosome in the 0-frame. Similar frame-shifting events could be the main cause of various phenotypes associated with environmental or genetically induced changes in the levels of aminoacylated tRNA.
Alleles↗
The transition state for formation of the peptide bond in the ribosome.
Using quantum mechanics and exploiting known crystallographic coordinates of tRNA substrate located in the ribosome peptidyl transferase center around the 2-fold axis, we have investigated the mechanism for peptide-bond formation. The calculation is based on a choice of 50 atoms assumed to be important in the mechanism. We used density functional theory to optimize the geometry and energy of the transition state (TS) for peptide-bond formation. The TS is formed simultaneously with the rotatory motion enabling the translocation of the A-site tRNA 3' end into the P site, and we estimated the magnitude of rotation angle between the A-site starting position and the place at which the TS occurs. The calculated TS activation energy, E(a), is 35.5 kcal (1 kcal = 4.18 kJ)/mol, and the increase in hydrogen bonding between the rotating A-site tRNA and ribosome nucleotides as the TS forms appears to stabilize it to a value qualitatively estimated to be approximately 18 kcal/mol. The optimized geometry corresponds to a structure in which the peptide bond is being formed as other bonds are being broken, in such a manner as to release the P-site tRNA so that it may exit as a free molecule and be replaced by the translocating A-site tRNA. At TS formation the 2' OH group of the P-site tRNA A76 forms a hydrogen bond with the oxygen atom of the carboxyl group of the amino acid attached to the A-site tRNA, which may be indicative of its catalytic role, consistent with recent biochemical experiments.
Crystallography, X-Ray↗
The C-terminal appended domain of human cytosolic leucyl-tRNA synthetase is indispensable in its interaction with arginyl-tRNA synthetase in the multi-tRNA synthetase complex.
Human cytosolic leucyl-tRNA synthetase is one component of a macromolecular aminoacyl-tRNA synthetase complex. This is unlike prokaryotic and lower eukaryotic LeuRSs that exist as free soluble enzymes. There is little known about it, since the purified enzyme has been unavailable. Herein, human cytosolic leucyl-tRNA synthetase was heterologously expressed in a baculovirus system and purified to homogeneity. The molecular mass (135 kDa) of the enzyme is close to the theoretical value derived from its cDNA. The kinetic constants of the enzyme for ATP, leucine, and tRNA(Leu) in the ATP-PP(i) exchange and tRNA leucylation reactions were determined, and the results showed that it is quite active as a free enzyme. Human cytosolic leucyl-tRNA synthetase expressed in human 293 T cells localizes predominantly to the cytosol. Additionally, it is found to have a long C-terminal extension that is absent from bacterial and yeast LeuRSs. A C-terminal 89-amino acid truncated human cytosolic leucyl-tRNA synthetase was constructed and purified, and the catalytic activities, thermal stability, and subcellular location were found to be almost identical to native enzyme. In vivo and in vitro experiments, however, show that the C-terminal extension of human cytosolic leucyl-tRNA synthetase is indispensable for its interaction with the N-terminal of human cytosolic arginyl-tRNA synthetase in the macromolecular complex. Our results also indicate that the two molecules interact with each other only through their appended domains.
Adenosine Triphosphate↗
Effect of spermine on the efficiency and fidelity of the codon-specific binding of tRNA to the ribosomes.
Binding of the yeast Tyr-tRNA and Phe-tRNA to the A site, and the binding of their acetyl derivatives to the P site of poly(U11,A)-programmed Escherichia coli ribosomes was studied. Spermine stimulated the rate of binding of both tRNAs at least threefold, enabling more than 90% final saturation of both ribosomal binding sites. The effect is observed when the tRNAs, but not ribosomes or poly(U11,A), are preincubated with polyamine. Regardless of the binding site, optimal saturation was reached at spermine/tRNA molar ratios of 3 for tRNA(Phe) and 5 for tRNA(Tyr). The same low spermine/tRNA ratios were previously reported to stabilize the conformation of these tRNAs in solution. On the other hand, the messenger-free, EF-Tu- and EF-G-dependent polymerization of lysine from E. coli Lys-tRNA is drastically reduced, while the poly(A)-directed polymerization is stimulated by spermine through a wide range of Mg2+ concentrations. Misreading of UUU codons as isoleucine, assayed by the A-site binding of E. coli Ile-tRNA, is also inhibited by spermine. All these results demonstrate that spermine increases the efficiency and accuracy of a series of macromolecular interactions leading to the correct incorporation of an amino acid into protein, at the same time preventing some unspecific or erroneous interactions. From the analogy with its known structural effects, it can be inferred that spermine does so by conferring on the tRNA a specific biologically functional conformation.
Binding Sites↗
Influence of temperature on tRNA modification in archaea: Methanococcoides burtonii (optimum growth temperature [Topt], 23 degrees C) and Stetteria hydrogenophila (Topt, 95 degrees C).
We report the first study of tRNA modification in psychrotolerant archaea, specifically in the archaeon Methanococcoides burtonii grown at 4 and 23 degrees C. For comparison, unfractionated tRNA from the archaeal hyperthermophile Stetteria hydrogenophila cultured at 93 degrees C was examined. Analysis of modified nucleosides using liquid chromatography-electrospray ionization mass spectrometry revealed striking differences in levels and identities of tRNA modifications between the two organisms. Although the modification levels in M. burtonii tRNA are the lowest in any organism of which we are aware, it contains more than one residue per tRNA molecule of dihydrouridine, a molecule associated with maintenance of polynucleotide flexibility at low temperatures. No differences in either identities or levels of modifications, including dihydrouridine, as a function of culture temperature were observed, in contrast to selected tRNA modifications previously reported for archaeal hyperthermophiles. By contrast, S. hydrogenophila tRNA was found to contain a remarkable structural diversity of 31 modified nucleosides, including nine methylated guanosines, with eight different nucleoside species methylated at O-2' of ribose, known to be an effective stabilizing motif in RNA. These results show that some aspects of tRNA modification in archaea are strongly associated with environmental temperature and support the thesis that posttranscriptional modification is a universal natural mechanism for control of RNA molecular structure that operates across a wide temperature range in archaea as well as bacteria.
Cell Division↗
The effect of pseudouridine and pH on the structure and dynamics of the anticodon stem-loop of tRNA(Lys,3).
The anticodon stem-loop hairpin of tRNA(Lys,3) was synthesized and the solution structure determined by NMR spectroscopy. The hairpin is thermodynamically stabilized by pseudouridine as determined by UV Tm measurements, and the local loop structure is stabilized with base-stacking of the nucleosides in the anticodon loop 5' of the psi 39 nucleoside modification. The tRNA(Lys,3) hairpin also contains an A(+)-C base-pair that effectively reduces the size of the normal 7 nucleotide anticodon loop to 5 nucleotides and induces a change in the loop backbone conformation. The stabilizing effects of the A(+)-C base-pair and pseudouridine are only partially additive, suggesting that the conformational changes caused by each modification are not completely compatible. The structure of the anticodon loop is distinctly different from that seen for other tRNAs exemplified by tRNA(Phe), suggesting that the full complement of modified nucleosides present in tRNA(Lys,3) should significantly change the structure compared to the unmodified tRNA anticodon loop. The conformation of the loop has important implications for the role of nucleoside modification in codon-anticodon recognition and for utilization of tRNA(Lys,3) by HIV-1 as the natural reverse transcriptase primer.
Anticodon↗
The context theory as applied to the decoding of the initiator tRNA by Escherichia coli ribosomes.
The involvement of nucleotides adjacent to the termination codons in tRNA during the suppression of termination has been formulated as the 'context theory' by Bossi and Roth (1980) [Nature (Lond.) 286, 123-127]. The finding that U-U-G functions as an initiator codon has revived the discussion on the participation of the nucleotides flanking the initiator triplet in the decoding of initiator tRNA (context theory of initiation by the ribosome). We compared the capacity of oligonucleotides cognate to the anticodon loop of formylmethionine tRNA, such as A-U-G, A-U-G-A and U-A-U-G-A, to enhance the formation of the 30-S and 70-S ribosomal initiation complexes. Three different methods were used to determine the apparent binding constants and the stoichiometries of the respective complexes: adsorption of the complexes to nitrocellulose filters, equilibrium dialysis, and velocity sedimentation. We found that in the 30-S ribosomal initiation complex and in the presence of initiation factor 2 and GTP, formylmethionyl-tRNA is preferentially decoded by more than three mRNA bases. With the 70-S ribosome, however, once initiation factor 2 had been released, A-U-G represented the most effective codon to direct the formylmethionyl-tRNA to the peptidyl site. An extended initiator sequence may either give additional stability to the 30-S initiation complex or may allow for an ambiguity by one base pair in the decoding of the initiator tRNA.
Binding Sites↗
Kirromycin drastically reduces the affinity of Escherichia coli elongation factor Tu for aminoacyl-tRNA.
We have studied the interaction between EF-Tu-GDP or EF-Tu-GTP in complex with kirromycin or aurodox (N1-methylkirromycin) and aminoacyl-tRNA, N-acetylaminoacyl-tRNA, or deacylated tRNA. Three independent methods were used: zone-interference gel electrophoresis, GTPase stimulation, and fluorescence. All three methods revealed that kirromycin induces a severe drop in the stability of the complex of EF-Tu-GTP and aminoacyl-tRNA of about 3 orders of magnitude. The affinities of EF-Tu-kirromycin-GTP and EF-Tu-kirromycin-GDP for aa-tRNA were found to be of about the same order of magnitude. We conclude that kirromycin and related compounds do not induce a so-called GTP-like conformation of EF-Tu with respect to tRNA binding. The findings shed new light on the mechanism of action of the antibiotic during the elongation cycle. In contrast to indirect evidence previously obtained in our laboratory [Van Noort et al. (1982) EMBO J. 1, 1199-1205; Van Noort et al. (1986) Proc. Natl. Acad. Sci. U.S.A. 71, 4910-4914], we were unable to demonstrate complexes of EF-Tu-aurodox-GTP/GDP with N-acetylaminoacyl-tRNA or deacylated tRNA by direct detection using zone-interference gel electrophoresis. Modification with N-tosyl-L-phenylalanine chloromethyl ketone (TPCK) decreases the affinity of EF-Tu-kirromycin-GTP for aminoacyl-tRNA, just like it does in the absence of the antibiotic.
Electrophoresis, Agar Gel↗
Magnesium ions are required by Bacillus subtilis ribonuclease P RNA for both binding and cleaving precursor tRNAAsp.
The multiple roles Mg2+ plays in ribozyme-catalyzed reactions in stabilizing RNA structure, enhancing the affinity of bound substrates, and increasing catalysis are delineated for the RNA component of ribonuclease P (RNase P RNA) by a combination of steady-state kinetics, transient kinetics, and equilibrium binding measurements. Divalent metal ions cooperatively increase the affinity of Bacillus subtilis RNase P RNA for B. subtilis tRNA(Asp) more than 10(3)-fold, consistent with at least two additional magnesium ions binding to the RNase P RNA.tRNA complex. Monovalent cations also decrease KD(tRNA) and reduce, but do not eliminate, the dependence on magnesium ions, demonstrating that nonspecific electrostatic shielding is not sufficient to explain the requirement for high salt. Both di- and monovalent cations promote the high affinity of tRNA by forming contacts in the binary complex that reduce the dissociation rate constant for tRNA. Additionally, the hyperbolic dependence of the hydrolytic rate constant on the concentration of magnesium with a K1/2 approximately equal to 36 mM suggests that a third low-affinity divalent metal ion stabilizes the transition state for pre-tRNA cleavage. Furthermore, many (about 100) magnesium ions bind independently to RNase P RNA with higher affinity than the K1/2 of any of the functionally characterized magnesium binding sites. Therefore, the magnesium binding sites that have differential affinity in either the "folded" species or binary complex are a small subset of the total number of associated magnesium ions. In summary, the importance of magnesium bound to RNase P RNA can be separated functionally into three crucial roles: at least three sites stabilize the folded RNA tertiary structure [Pan. T. (1995) Biochemistry 34, 902-909], at least two sites enhance the formation of complexes of RNase P RNA with pre-tRNA or tRNA, and at least one site stabilizes the transition state for pre-tRNA cleavage.
Bacillus subtilis↗
Purification and characterization of a tRNA methylase from Salmonella typhimurium.
A tRNA methylase, in which supK strains of Salmonella typhimurium are deficient, was purified from strain LT2 and characterized. Column chromatography of protein extracts from wild-type cells on phosphocellulose, diethylaminoethyl-Sephadex A-50, and hydroxlapatite resulted in an enzyme that was estimated to be about 50% pure. tRNA from S. typhimurium which had been incubated at pH 9.0 served as a substrate for this methylase. The enzyme has a molecular weight of about 50,000 as estimated by gel chromatography and by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels. The optimal assay conditions, as well as the kinetics and stability of the enzyme, were studied. As with other tRNA-methylating enzymes, S-adenosylhomocysteine is a potent inhibitor.
Kinetics↗
Regulated translation termination at the upstream open reading frame in s-adenosylmethionine decarboxylase mRNA.
The upstream open reading frame (uORF) in the mRNA encoding S-adenosylmethionine decarboxylase is a cis-acting element that confers feedback control by cellular polyamines on translation of this message. Recent studies demonstrated that elevated polyamines inhibit synthesis of the peptide encoded by the uORF by stabilizing a ribosome paused in the vicinity of the termination codon. These studies suggested that polyamines act at the termination step of uORF translation. In this paper, we demonstrate that elevated polyamines stabilize an intermediate in the termination process, the complete nascent peptide linked to the tRNA that decodes the final codon. The peptidyl-tRNA molecule is found associated with the ribosome fraction, and decay of this molecule correlated with release of the paused ribosome from the message. Furthermore, the stability of this complex is influenced by the same parameters that influence regulation by the uORF in vivo, namely the concentration of polyamines and the sequence of the uORF-encoded peptide. These results suggest that the regulated step in uORF translation is after formation of the peptidyl-tRNA molecule but before hydrolysis of the peptidyl-tRNA bond. This regulation may involve an interaction between the peptide, polyamines, and a target in the translational apparatus.
Adenosylmethionine Decarboxylase↗
Use of a gene encoding a suppressor tRNA as a reporter of transcription: analyzing the action of the Nun protein of bacteriophage HK022.
The Nun protein of phage HK022 blocks the expression of genes that lie downstream of the nut sites of phage lambda. Nun is believed to act by promoting premature termination of transcription at or near these sites. To test this hypothesis and to facilitate mapping the sites of termination, we inserted a gene encoding a suppressor tRNA immediately downstream of the lambda nutL site and determined the effect of Nun on tRNA level. We found that Nun severely reduced the accumulation of mature, biologically active tRNA and promoted the accumulation of short, promoter-proximal transcripts whose 3' ends were dispersed over a 100-nucleotide region downstream of nutL. These results are consistent with the hypothesis that Nun terminates transcription within the region immediately downstream of nutL and are inconsistent with the hypothesis that the only action of Nun is to prevent translation of genes located downstream of the nut site. The stability, small size, and easily assayable biological function of suppressor tRNA recommend it as a reporter of transcription in other systems.
Bacteriophage lambda↗