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PubMed1972

Structure and function of Escherichia coli formylmethionine transfer RNA: loss of methionine acceptor activity by modification of a specific guanosine residue in the acceptor stem of formylmethionine transfer RNA from Escherichia coli.

The structural requirements of E. coli formylmethionine tRNA for aminoacylation have been examined by chemical modification of the tRNA, followed by separation of the modified molecules into active and inactive components. Photooxidation of tRNA(fMet) at 50 degrees in the presence of methylene blue results in modification of two guanosine (G) residues in the acceptor stem, at positions no. 2 and no. 71 from the 5'-phosphate terminus. Both of these modifications are present in inactive molecules, but only the G residue at position no. 2 is modified in the acceptor stem of active molecules. Loss of methionine acceptance occurs with first-order kinetics, indicating that inactivation by modification of G residue no. 71 is independent of any other modifications taking place under these conditions. The presence of a modified G residue at position no. 2 in the acceptor stem of active photooxidized molecules shows that disruption of normal base-pairing in this region is not sufficient to inactivate tRNA(fMet). These data indicate that the inactivating modification at position no. 71 is lethal due to a specific alteration in the nucleotide base, rather than simply as a result of breaking a hydrogen-bonded base pair in the acceptor stem.

Acylation↗
PubMed1994

Rit1, a tRNA backbone-modifying enzyme that mediates initiator and elongator tRNA discrimination.

Using a genetic screen in yeast aimed at identifying cellular factors involved in initiator and elongator methionine tRNA discrimination in the translational process, we have identified a mutation that abolish the requirement for elongator methionine tRNA. The gene affected, which we call the ribosylation of the initiator tRNA gene or RIT1, encodes a 2'-O-ribosyl phosphate transferase. This enzyme modifies exclusively the initiator tRNA in position 64 using 5'-phosphoribosyl-1'-pyrophosphate as the modification donor. As the initiator tRNA participates both in the initiation and elongation of translation in a rit1 strain, we conclude that the 2'-O-ribosyl phosphate modification discriminates the initiator tRNAs from the elongator tRNAs during protein synthesis. The modification enzyme was shown to recognize the stem-loop IV region that is unique in eukaryotic cytoplasmic initiator tRNAs.

Alleles↗
PubMed1993

Modified nucleotides in Bacillus subtilis tRNA(Trp) hyperexpressed in Escherichia coli.

In the present study, modified nucleotides in the B. subtilis tRNA(Trp) cloned and hyperexpressed in E. coli have been identified by TLC and HPLC analyses. The modification patterns of the two isoacceptors of cloned B. subtilis tRNA(Trp) have been compared with those of native tRNA(Trp) from B. subtilis and from E. coli. The modifications of the A73 mutant of B. subtilis tRNA(Trp), which is inactive toward its cognate TrpRS, were also investigated. The results indicate the formation of the modified nucleotides S4U8, Gm18, D20, Cm32, i6A/ms2i6A37, T54 and psi 55 on cloned B. subtilis tRNA(Trp). This modification pattern resembles the pattern of E. coli tRNA(Trp), except that m7G is missing from the cloned tRNA(Trp), probably on account of its short extra loop. In contrast, the pattern departs substantially from that of native B. subtilis tRNA(Trp). Therefore, the cloned B. subtilis tRNA(Trp) has taken on largely the modification pattern of E. coli tRNA(Trp) despite the 26% sequence difference between the two species of tRNA, gaining in particular the Cm32 and Gm18 modifications from the E. coli host. A notable difference between the isoacceptors of the cloned tRNA(Trp) was seen in the extent of modification of A37, which occurred as either the hypomodified i6A or the hypermodified ms2i6A form. Surprisingly, base substitution of guanosine by adenosine at position 73 of the cloned tRNA(Trp) has led to the abolition of the 2'-O-methylation modification of the remote G18 residue.

Bacillus subtilis↗
PubMed2003

The GTPase activity and C-terminal cysteine of the Escherichia coli MnmE protein are essential for its tRNA modifying function.

The Escherichia coli MnmE protein is a three-domain protein that exhibits a very high intrinsic GTPase activity and low affinity for GTP and GDP. The middle GTPase domain, when isolated, conserves the high intrinsic GTPase activity of the entire protein, and the C-terminal domain contains the only cysteine residue present in the molecule. MnmE is an evolutionarily conserved protein that, in E. coli, has been shown to control the modification of the uridine at the wobble position of certain tRNAs. Here we examine the biochemical and functional consequences of altering amino acid residues within conserved motifs of the GTPase and C-terminal domains of MnmE. Our results indicate that both domains are essential for the MnmE tRNA modifying function, which requires effective hydrolysis of GTP. Thus, it is shown for the first time that a confirmed defect in the GTP hydrolase activity of MnmE results in the lack of its tRNA modifying function. Moreover, the mutational analysis of the GTPase domain indicates that MnmE is closer to classical GTPases than to GTP-specific metabolic enzymes. Therefore, we propose that MnmE uses a conformational change associated with GTP hydrolysis to promote the tRNA modification reaction, in which the C-terminal Cys may function as a catalytic residue. We demonstrate that point mutations abolishing the tRNA modifying function of MnmE confer synthetic lethality, which stresses the importance of this function in the mRNA decoding process.

Bacterial Proteins↗
PubMedDate not supplied

Structure and function of ribosomal RNA.

A refined model has been developed for the folding of 16S rRNA in the 30S subunit, based on additional constraints obtained from new experimental approaches. One set of constraints comes from hydroxyl radical footprinting of each of the individual 30S ribosomal proteins, using free Fe(2+)-EDTA complex. A second approach uses localized hydroxyl radical cleavage from a single Fe2+ tethered to unique positions on the surface of single proteins in the 30S subunit. This has been carried out for one position on the surface of protein S4, two on S17, and three on S5. Nucleotides in 16S rRNA that are essential for P-site tRNA binding were identified by a modification interference strategy. Ribosomal subunits were partially inactivated by chemical modification at a low level. Active, partially modified subunits were separated from inactive ones by binding 3'-biotinderivatized tRNA to the 30S subunits and captured with streptavidin beads. Essential bases are those that are unmodified in the active population but modified in the total population. The four essential bases, G926, 2mG966, G1338, and G1401 are a subset of those that are protected from modification by P-site tRNA. They are all located in the cleft of our 30S subunit model. The rRNA neighborhood of the acceptor end of tRNA was probed by hydroxyl radical probing from Fe2+ tethered to the 5' end of tRNA via an EDTA linker. Cleavage was detected in domains IV, V, and VI of 23S rRNA, but not in 5S or 16S rRNA. The sites were all found to be near bases that were protected from modification by the CCA end of tRNA in earlier experiments, except for a set of E-site cleavages in domain IV and a set of A-site cleavages in the alpha-sarcin loop of domain VI. In vitro genetics was used to demonstrate a base-pairing interaction between tRNA and 23S rRNA. Mutations were introduced at positions C74 and C75 of tRNA and positions 2252 and 2253 of 23S rRNA. Interaction of the CCA end of tRNA with mutant ribosomes was tested using chemical probing in conjunction with allele-specific primer extension. The interaction occurred only when there was a Watson-Crick pairing relationship between positions 74 of tRNA and 2252 of 23S rRNA. Using a novel chimeric in vitro reconstitution method, it was shown that the peptidyl transferase reaction depends on this same Watson-Crick base pair.

Models, Biological↗
PubMed1987

Binding stoichiometry of tRNATrp and tryptophanyl-tRNA synthetase from bovine pancreas under pH conditions of maximum activity. Analysis by ultracentrifugation, fluorescence quenching and chemical modification.

The binding stoichiometry of tRNATrp and tryptophanyl-tRNA synthetase (EC 6.1.1.2) from beef is examined by three approaches, under pH conditions of maximum activity (pH 8.0). (1) Analytical ultracentrifugation evidences the binding of a single mol of tRNATrp in a 2.5-10 microM concentration range. (2) tRNATrp quenches the fluorescence of the enzyme. The dependence of this fluorescence quenching on the tRNATrp concentration (0.1-4 microM) reflects also the binding of 1 mol of tRNA per mol of enzyme, with a Kd value of 0.19 +/- 0.02 microM. (3) tRNATrp protects the enzyme against derivatization by oxidized ATP. Out of the two fast-reacting lysine residues of the native enzyme, only one is prevented from reacting by tRNATrp in the 0.5-110 microM concentration range. This protection can be significantly analyzed only by assuming a one-to-one complex between the enzyme and tRNA. These results, obtained at pH 8.0 and 25 degrees C, are in contrast with the stoichiometry of 2 mol of tRNA to 1 mol of enzyme, previously observed at pH 6.0 and 4 degrees C.

Amino Acyl-tRNA Synthetases↗
PubMed1983

Incorporation of 3H-amino acids into proteins in a partially purified fraction of axoplasm: evidence for transfer RNA-mediated, post-translational protein modification in squid giant axons.

Transfer RNA (tRNA) has been demonstrated to be present in axons of both invertebrates and the higher vertebrates, but nothing is known of its role in the metabolism of the axon. The present experiments were performed to determine whether tRNA functions in axons as a participant in post-translational protein modification of endogenous proteins. RNA was extracted from the axoplasm of squid giant axons and incubated with a variety of 3H-amino acids, aminoacyl-tRNA synthetases (obtained from squid optic lobe), and an appropriate reaction mixture. All of the amino acids tested were bound to an RNA fraction, but this reaction did not occur when samples were incubated in the presence of ribonuclease or in the absence of axoplasmic RNA. When radioactive RNA was chromatographed by polyacrylamide gel electrophoresis, the radioactivity comigrated with known tRNA markers, suggesting the presence of 3H-aminoacylated tRNA. Aminoacylation of RNA could also be demonstrated by incubating fresh axoplasm with labeled amino acids and a reaction mixture, minus exogenous aminoacyl-tRNA synthetases. These findings indicate the presence in axoplasm of a variety of species of aminoacyl-tRNAs as well as their corresponding synthetase enzymes. In the latter experiment no radioactivity was found associated with the protein fraction. This was also the finding when 3H-aminoacylated tRNA was either injected directly into the axon or incubated with extruded axoplasm. Thus, under the conditions described above, there is no evidence of transfer of amino acids from tRNA to proteins. In other experiments, axoplasm was pooled to a volume of 50 to 100 microliters, homogenized gently, and centrifuged at 150,000 X g for 1 hr. Some of the high speed supernatant was incubated with labeled amino acids and an appropriate reaction mixture, and the remainder was passed through an S-200 Sephacryl column before incubation with the same reaction mixture. There was no incorporation of amino acids into protein in the high speed supernatant fraction. However, in the S-200 purified fraction 3H-labeled Arg, Lys, Tyr, Leu, and Asp were all incorporated into proteins in amounts of 44, 30, 7, 5 and 3.5 times heat-inactivated controls. The reaction is not inhibited by Ca2+ or Ca2+-activated proteases, but appears to be dependent on the presence of tRNA. The addition of amino acids to protein is not protein synthesis since the reactions occurred in a partially purified fraction of the 150,000 X g supernatant, a fraction devoid of ribosomes and free amino acids.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acids↗
PubMed1975

Alterations in SVT2 cell transfer RNAs in response to cell density and serum type.

The chromatographic elution profiles of tRNA-A sn, tRNA-A sp, tRNA-H is and tRNA-T y r from SV40-transformed BALB-3T3 cells grown in fetal calf serum or cald serum-supplemented media have been examined. The relative proportions of certain of the isoaccepting species of these four tRNAs are altered in a similar fashion depending on the serum type. It is suggested that the elution profile alterations reflect the extent of modifications of a specific G residue to the minor nucleoside Q, and that this process differs between untransformed and transformed cells. In addition, cell density appears to influence the Q content of these tRNAs, though other density-dependent tRNA modifications also appear to occur.

Animals↗
PubMed1976

The methylation of tRNA.

The methylation of tRNA is a post-transcriptional modification which is achieved by specific enzymes, the tRNA methylases, with S adenosylmethionine as a methyl donor. The level and pattern of methylation are characteristic of the tRNA species and origin. Abnormally methylated tRNAs have been obtained, in vivo and in vitro, by a variety of methods, and their properties have been studied. The tRNA methylases are found in all cells and tissues. Their activity varies with the differentiation state of the cells, and under the influence of many internal and external factors ; it is especially elevated in embryonic and cancerous tissues. These enzymes are very unstable, and none of them has been purified to homogeneity. We present here their known properties and we propose a theory concerning their specificity. Finally, after reviewing the few available experimental data, we discuss the current hypotheses and speculations about the roles and functions of tRNA methylation.

Animals↗
PubMed1983

Comparison of Escherichia coli tRNAPhe in the free state, in the ternary complex and in the ribosomal A and P sites by chemical probing.

tRNAPheE.coli was modified at accessible guanosine, cytidine, and adenosine residues using the chemical modification method described by Peattie and Gilbert [Proc. Natl Acad. Sci. USA, 77, 4679-4689 (1980)]. Modification characteristics of the tRNA in the free state, in the ternary complex with elongation factor EF-Tu and GTP and in the ribosomal A and P sites were compared. A special procedure was devised to monitor, exclusively, tRNA molecules in the aminoacylated state. In the free tRNA, the most reactive bases are confined to the A73-C-C-A sequence of the aminoacyl stem, the anticodon loop, the D-loop and the extra loop and the results correlate well with the three-dimensional structure of tRNAPheyeast determined by X-ray studies. The pattern of reactivity was not affected either by charging the tRNA with phenylalanine or by labelling the 3' terminus with pCp. In the ternary complex, with elongation factor EF-Tu and GTP, changes in modification were observed at two sites, A73-C-C-A at the 3' terminus and C-13 and C-17 in the D-loop region, which are about 6 nm apart; no difference was observed in the anticodon loop. tRNAPhe bound at the ribosomal A or P sites exhibited similar, but not identical, modification patterns. Whereas nucleotides C-74 and C-75 were strongly protected at both sites, the adjacent A-73 showed an enhanced reactivity in the A site. The anticodon region G34-A-A-ms2.6(1)A was also strongly protected at both sites. In addition, nucleotide A-21 was protected during A-site, but not P-site, binding.

Binding Sites↗
PubMed1994

Differences in the levels of aminoacylation and contents of modified nucleotides between total tRNAs from N2- and NH4(+)-grown Azospirillum lipoferum cells.

Total tRNAs isolated from N2- and NH4(+)-grown Azospirillum lipoferum cells were compared with respect to amino acid acceptance, isoacceptor tRNA species levels and extent of nucleotide modifications. Amino-acylation of these two tRNA preparations with ten different amino acids indicated differences in the relative acceptor activities. Comparison of aminoacyl-tRNA patterns by RPC-5 column chromatography revealed no qualitative differences in the elution profiles. However, quantitative differences in the relative amounts of some isoacceptors were observed. These results indicate that alterations of relative amounts of functional tRNA species occur to match cellular requirements of the bacterial cells using N2 or NH4+ as nitrogen source. In addition, the content of modified nucleotides in total tRNAs of N2- and NH4(+)-grown cells was determined. In the NH4(+)-grown cells, content of most of the modified nucleotides decreased significantly. Based upon these results, the relationship of chargeability of tRNAs to base modifications is discussed.

Acylation↗
PubMed1998

A pseudoknotted tRNA variant is a substrate for tRNA (cytosine-5)-methyltransferase from Xenopus laevis.

tRNA post-transcriptional modification enzymes of Xenopus laevis were proposed previously to belong to two major groups according to their sensitivity to structural perturbations in their substrates. To further investigate the structural variations tolerated by these enzymes, the tRNA-like domain of turnip yellow mosaic virus RNA (88 nucleotides in length) has been microinjected into the oocytes of Xenopus laevis. This RNA possesses 12 potential target nucleotides for modification within a structure including a pseudoknotted folding, an extended anticodon stem, and unusual D-loop/T-loop interactions. Results indicate that only cytosine-42, a position equivalent to C-49 in canonical tRNAs, was quantitatively modified into m5C in the microinjected RNA. Modification was detected to high levels, indicating that at least one enzyme tolerates non-canonical structural features.

Animals↗
PubMed1985

Yeast tRNAAsp tertiary structure in solution and areas of interaction of the tRNA with aspartyl-tRNA synthetase. A comparative study of the yeast phenylalanine system by phosphate alkylation experiments with ethylnitrosourea.

Ethylnitrosourea is an alkylating reagent preferentially modifying phosphate groups in nucleic acids. It was used to monitor the tertiary structure, in solution, of yeast tRNAAsp and to determine those phosphate groups in contact with the cognate aspartyl-tRNA synthetase. Experiments involve 3' or 5'-end-labelled tRNA molecules, low yield modification of the free or complexed nucleic acid and specific splitting at the modified phosphate groups. The resulting end-labelled oligonucleotides are resolved on polyacrylamide sequencing gels and data analysed by autoradiography and densitometry. Experiments were conducted in parallel on yeast tRNAAsp and on tRNAPhe. In that way it was possible to compare the solution structure of two elongator tRNAs and to interpret the modification data using the known crystal structures of both tRNAs. Mapping of the phosphates in free tRNAAsp and tRNAPhe allowed the detection of differential reactivities for phosphates 8, 18, 19, 20, 22, 23, 24 and 49: phosphates 18, 19, 23, 24 and 49 are more reactive in tRNAAsp, while phosphates 8, 20 and 22 are more reactive in tRNAPhe. All other phosphates display similar reactivities in both tRNAs, in particular phosphate 60 in the T-loop, which is strongly protected. Most of these data are explained by the crystal structures of the tRNAs. Thermal transitions in tRNAAsp could be followed by chemical modifications of phosphates. Results indicate that the D-arm is more flexible than the T-loop. The phosphates in yeast tRNAAsp in contact with aspartyl-tRNA synthetase are essentially contained in three continuous stretches, including those at the corner of the amino acid accepting and D-arm, at the 5' side of the acceptor stem and in the variable loop. When represented in the three-dimensional structure of the tRNAAsp, it clearly appears that one side of the L-shaped tRNA molecule, that comprising the variable loop, is in contact with aspartyl-tRNA synthetase. In yeast tRNAPhe interacting with phenylalanyl-tRNA synthetase, the distribution of protected phosphates is different, although phosphates in the anticodon stem and variable loop are involved in both systems. With tRNAPhe, the data cannot be accommodated by the interaction model found for tRNAAsp, but they are consistent with the diagonal side model proposed by Rich & Schimmel (1977). The existence of different interaction schemes between tRNAs and aminoacyl-tRNA synthetases, correlated with the oligomeric structure of the enzyme, is proposed.

Alkylation↗
PubMed1996

The 2'-5' RNA ligase of Escherichia coli. Purification, cloning, and genomic disruption.

An RNA ligase previously detected in extracts of Escherichia coli is capable of joining Saccharomyces cerevisiae tRNA splicing intermediates in the absence of ATP to form a 2'-5' phosphodiester linkage (Greer, C., Javor, B., and Abelson, J. (1983) Cell 33, 899-906). This enzyme specifically ligates tRNA half-molecules containing nucleoside base modifications and shows a preference among different tRNA species. In order to investigate the function of this enzyme in RNA metabolism, the ligase was purified to homogeneity from E. coli lysate utilizing chromatographic techniques and separation of proteins by SDS-polyacrylamide gel electrophoresis. A single polypeptide of approximately 20 kilodaltons exhibited RNA ligase activity. The amino terminus of this protein was sequenced, and the open reading frame (ORF) encoding it was identified by a data base search. This ORF, which encodes a novel protein with a predicted molecular mass of 19.9 kDa, was amplified from E. coli genomic DNA and cloned. ORFs coding for highly similar proteins were detected in Methanococcus jannaschii and Bacillus stearothermophilus. The chromosomal gene encoding RNA ligase in E. coli was disrupted, abolishing ligase activity in cell lysates. Cells lacking ligase activity grew normally under laboratory conditions. However, moderate overexpression of the ligase protein led to slower growth rates and a temperature-sensitive phenotype in both wild-type and RNA ligase knockout strains. The RNA ligase reaction was studied in vitro using purified enzyme and was found to be reversible, indicating that this enzyme may perform cleavage or ligation in vivo.

Amino Acid Sequence↗
PubMed1985

Effect of trypsin modification of the Escherichia coli elongation factor Tu on the ternary complex with aminoacyl-tRNA.

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.

Amino Acid Sequence↗
PubMed1992

Pseudouridine in the anticodon G psi A of plant cytoplasmic tRNA(Tyr) is required for UAG and UAA suppression in the TMV-specific context.

We have previously isolated and sequenced Nicotiana cytoplasmic tRNA(Tyr) with G psi A anticodon which promotes readthrough over the leaky UAG termination codon at the end of the 126 K cistron of tobacco mosaic virus RNA and we have demonstrated that tRNA(Tyr) with Q psi A anticodon is no UAG suppressor. Here we show that the nucleotide in the middle of the anticodon (i.e., psi 35) also contributes to the suppressor efficiency displayed by cytoplasmic tRNA(Tyr). A tRNA(Tyr) with GUA anticodon was synthesized in vitro using T7 RNA polymerase transcription. This tRNA(Tyr) was unable to suppress the UAG codon, indicating that nucleotide modifications in the anticodon of tRNA(Tyr) have either stimulating (i.e., psi 35) or inhibitory (i.e., Q34) effects on suppressor activity. Furthermore, we have shown that the UAA but not the UGA stop codon is also efficiently recognized by tobacco tRNA(G psi ATyr), if placed in the TMV context. Hence this is the first naturally occurring tRNA for which UAA suppressor activity has been demonstrated. In order to study the influence of neighbouring nucleotides on the readthrough capacity of tRNA(Tyr), we have established a system, in which part of the sequence around the leaky UAG codon of TMV RNA was inserted into a zein pseudogene which naturally harbours an UAG codon in the middle of the gene. The construct was cloned into the vector pSP65 and in vitro transcripts, generated by SP6 RNA polymerase, were translated in a wheat germ extract depleted of endogenous mRNAs and tRNAs. A number of mutations in the codons flanking the UAG were introduced by site-directed mutagenesis. It was found that changes at specific positions of the two downstream codons completely abolished the readthrough over the UAG by Nicotiana tRNA(Tyr), indicating that this tRNA needs a very specific codon context for its suppressor activity.

Anticodon↗
PubMed1990

Protection from chemical modification of nucleotides in complexes of M1 RNA, the catalytic subunit of RNase P from E coli, and tRNA precursors.

Certain nucleotides in M1 RNA, the catalytic RNA subunit of RNase P from E coli, are protected from chemical modification when M1 RNA forms complexes with tRNA precursor molecules (ES complexes). Many of these nucleotides are important in the formation of the Michaelis complex. In the presence of tRNA precursor molecules, the pattern of protection from chemical modification of a region in M1 RNA that resembles the E site in 23S rRNA is similar to the pattern of protection of the E site in the presence of deacylated tRNA. In the complex with the RNA enzyme, more nucleotides in the substrate become accessible to modification, an indication that the substrate is in an unfolded conformation under these conditions.

Base Sequence↗