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PubMed1990

Inhibition of the p66/p51 form of human immunodeficiency virus reverse transcriptase by tRNA(Lys).

Human immunodeficiency virus (HIV) reverse transcriptase (RT) uses host tRNA(Lys) partially annealed to the primer binding site (PBS) as primer for the initiation of cDNA synthesis. When assaying cDNA synthesis with a template-primer complex formed by an RNA fragment carrying the PBS site and bovine tRNA(Lys) we noticed that an excess of primer tRNA inhibited strongly the DNA polymerase activity of a recombinant HIV RT (p66-p51 heterodimeric form) produced in transformed yeast cells. The same inhibitory effect was observed with animal DNA polymerase alpha, while avian retrovirus RT was neither affected by tRNA(Lys) nor by its specific primer tRNA(Trp). Although the strongest inhibition was observed with tRNA(Lys), other tRNas like tRNA(Phe) and tRNA(Trp) inhibited also the HIV RT, whereas tRNAs specific for valine, proline and glycine had no effect on enzyme activity. Digestion of tRNA(Lys) with pancreatic RNase abolished the inhibition; on the other hand T1 RNase digestion had no effect on the inhibition suggesting a role of the anticodon region in this effect. The 12- and 14-mers corresponding to the anticodon regions of the three bovine tRNA(Lys) isoacceptors inhibited RT activity, indicating that at least an important part of the inhibitory effect could be ascribed to this tRNA region. A strong stimulation of DNA polymerase activity was observed when the effect of tRNA(Lys) was assayed on a recombinant HIV reverse transcriptase produced in a protease deficient yeast strain, which leads to the production of an active p66 enzyme. The same tRNAs that inhibited strongly the heterodimeric form stimulated the p66 form of HIV reverse transcriptase. The results suggest that although both enzymatic forms are able to interact with tRNA(Lys) the topography, as well as the functional implications of the interaction between the precursor and the mature form of HIV reverse transcriptase with the tRNA(Lys) primer, are different.

Base Sequence↗
PubMed1996

Limited proteolysis and amino acid replacements in the effector region of Thermus thermophilus elongation factor Tu.

The effector region of the elongation factor Tu (EF-Tu) from Thermus thermophilus was modified by limited proteolysis or via site-directed mutagenesis. The biochemical properties of the obtained EF-Tu variants were investigated with respect to partial reactions of the functional cycle of EF-Tu. EF-Tu that was cleaved at the Arg59-Gly60 peptide bond [EF-Tu-(1-59)/EF-Tu-(60-405)] bound GDP, EF-Ts and aminoacyl-tRNA, had normal intrinsic GTPase activity and was active in poly(U)-dependent poly(Phe) synthesis. However, the GTPase activity of EF-Tu-(1-59)/EF-Tu-(60-405) was not stimulated by T. thermophilus 70S ribosomes, and its GTP-dissociation rate was increased compared with that of intact EF-Tu. EF-Tu cleaved at the Lys52-Ala53 peptide bond has properties similar to EF-Tu-(1-59)/EF-Tu-(60-405). By means of site-directed mutagenesis, Glu55 was replaced by Leu, Glu56 by Ala and Arg59 by Thr in T. thermophilus EF-Tu. These amino acid substitutions did not substantially affect either the affinity of EF-Tu. GTP for aminoacyl-tRNA or the interactions with GDP, GTP or EF-Ts. Similarly the intrinsic GTPase activity is not influenced. Replacement of Glu56 by Ala led to strong reduction in the ribosome-induced GTPase activity. This effect is specific since replacement of the neighbouring Glu55 by Leu did not affect the ribosome-induced GTPase activity. The results demonstrate that the structure of the effector region of EF-Tu in the vicinity of Arg59 is important for the control of the GTPase activity by ribosomes.

Amino Acid Sequence↗
PubMedDate not supplied

[Temperature-dependent structural changes in complexes of tRNA(adenine-1)-methyltransferase from Thermus thermophilus HB8 and tRNA studied by optical and electron microscope methods].

The complexation of tRNA (adenine-1-)-methyltransferase from Thermus thermophilus HB8 (E.C.2.1.1.36) with Escherichia coli tRNA(Phe) and yeast tRNA1(Val) was investigated in a temperature range from 20 to 90 degrees C. The quantity of methylase subunits bounded with tRNA and the association constant (Ka) were determined by means of fluorescence quenching of the enzyme tryptophane residues by tRNA molecules. The number of enzyme subunits bounded with one tRNA molecule at temperatures 20-70 degrees C is equal to 8 +/- 2. The Ka values increase from (2 divided by 3).10(7) at 20 degrees C up to 8.5.10(7) M-1 at 70 degrees C. The temperature increase from 70 to 90 degrees C causes a decrease in the enzyme specific activity and in Ka values. In the temperature range from 75 to 90 degrees C a cooperative transition of methylase macromolecules into associates was observed. This association is accompanied by an increase of UV-light scattering and of fluorescence polarization coefficient of methylase tryptophane residues. In the absence of tRNA the size of enzyme associates (d) is evaluated to be more than 320 nm (d greater than or equal to lambda-320 nm), in the presence of tRNA-less than 320 nm (d much less than lambda-320 nm). An electron microscopic investigation of methylase and its complexes with tRNA at 20 degrees C revealed disk-like particles with a diameter and height of 8-11 nm and 4-5 nm, respectively. These disk-like methylase preparations dialized against distilled water form flexible polymeric rods with a diameter of 10-12 nm and the length of about several hundreds nm. During complexation of methylase with tRNA, in the same conditions as the dializes was carried out, large associates were not revealed.

Escherichia coli↗
PubMed2001

Identification of critical elements in the tRNA acceptor stem and T(Psi)C loop necessary for human immunodeficiency virus type 1 infectivity.

A mutant human immunodeficiency virus type 1 (HIV-1) with a primer binding site (PBS) complementary to yeast tRNA(Phe) (psHIV-Phe), which relies on exogenous yeast tRNA(Phe) as reverse transcription primer, was used to investigate elements in the tRNA acceptor stem and T(Psi)C stem-loop required for the tRNA primer selection and use in HIV-1 replication. tRNA(Phe) mutants with two- or four-nucleotide deletions in the 3' end retained the capacity to complement replication of psHIV-Phe. tRNA(Phe) mutants with an extended 5' end had reduced capacity for complementation, which could be restored by extension of the 3' end of these tRNA(Phe) mutants with sequences complementary to the HIV-1 U5 region. Further analysis of mutations in the acceptor stem of tRNA(Phe) suggested that an intact acceptor stem RNA structure is important for complementation. Analysis of single-nucleotide changes in the T(Psi)C stem-loop of tRNA(Phe) revealed an unexpected, essential role of this region for rescue of psHIV-Phe.

Binding Sites↗
PubMed2000

Essential regions of the tRNA primer required for HIV-1 infectivity.

Human immunodeficiency virus (HIV), like all retroviruses, requires a cellular tRNA as a primer for initiation of reverse transcription. In a previous study, we demonstrated that an HIV-1 with a primer binding site complementary to yeast tRNA(Phe) (psHIV-Phe) was not infectious unless yeast tRNA(Phe) was supplied in trans. This unique in vivo complementation system has now been used to define the elements of the tRNA required for HIV-1 replication. Mutant tRNA(Phe) with deletions in TPsiC stem-loop, anticodon stem-loop or D stem-loop of the tRNA were generated and assessed for the capacity to rescue psHIV-Phe. Mutant tRNA(Phe) with disrupted TPsiC stem-loop did not rescue psHIV-Phe. In contrast, a mutant tRNA(Phe) without the D stem-loop was fully functional for the rescue. The tRNA anticodon stem-loop region was found to be important for efficient complementation. The results of our studies demonstrate for the first time the importance of specific structural and sequence elements of the tRNA primer for HIV-1 reverse transcription and define new targets for interruption of HIV-1 replication.

Base Sequence↗
PubMed1996

Efficient modification of RNA by porphyrin cation photochemistry: monitoring the folding of coaxially stacked RNA helices in tRNA(Phe) and the human immunodeficiency virus type 1 rev response element RNA.

Coaxially stacked RNA helices are a determined of RNA tertiary structure, but their presence is rarely detected using conventional chemical modification methods. In this report we describe a porphyrin ion photoreaction that enables one to monitor RNA stacking interactions and the folding of coaxially stacked RNA helices. The porphyrin cations meso-tetrakis(4-N-methylpyridyl)porphine, meso-tetrakis-(para-N-trimethylanilinium)porphine, and meso-tetrakis(2-N-methylpyridyl)porphine were used to characterize tRNA(Phe) and the human immunodeficiency virus type-I Rev response element RNA. Nucleosides at the bases of contiguous RNA helices in each RNA are efficiently modified by the porphyrin cations following irradiation of porphyrin-RNA mixtures. These photomodifications are markedly reduced for RNA equilibrated in ionic buffers that lead to enhanced stabilization of coaxially stacked helices. The porphyrin cation photoreaction specifically modifies G18, G20, and G34 in the tRNA folding produced by Mg(II). These nucleobases are exposed to solvent in the native tRNA structure and thus available to stack with solvent-borne porphyrin molecules. The describe porphyrin cation photochemical method provides a novel approach to study the solvent accessibility of nucleobases in RNA structure and to monitor the folding of coaxially stacked helices in RNA.

Aniline Compounds↗
PubMed1992

Evidence for RNA in the peptidyl transferase center of Escherichia coli ribosomes as indicated by fluorescence.

A coumarin derivative was covalently attached to either the amino acid or the 5' end of phenylalanine-specific transfer RNA (tRNA(phe)). Its fluorescence was quenched by methyl viologen when the tRNA was free in solution or bound to Escherichia coli ribosomes. Methyl viologen as a cation in solution has a strong affinity for the ionized phosphates of a nucleic acid and so can be used to qualitatively measure the presence of RNA in the immediate vicinity of the tRNA-linked coumarins upon binding to ribosomes. Fluorescence lifetime measurements indicate that the increase in fluorescence quenching observed when the tRNAs are bound into the peptidyl site of ribosomes is due to static quenching by methyl viologen bound to RNA in the immediate vicinity of the fluorophore. The data lead to the conclusion that the ribosome peptidyl transferase center is rich in ribosomal RNA. Movement of the fluorophore at the N-terminus of the nascent peptide as it is extended or movement of the tRNA acceptor stem away from the peptidyl transferase center during peptide bond formation appears to result in movement of the probe into a region containing less rRNA.

Escherichia coli↗
PubMed2000

Evaluation of uranyl photocleavage as a probe to monitor ion binding and flexibility in RNAs.

In order to evaluate uranyl photocleavage as a tool to identify and characterize structural and dynamic properties in RNA, we compared uranyl cleavage sites in five RNA molecules with known X-ray structures, namely the hammerhead and hepatitis delta virus ribozymes, the P4-P6 domain of the Tetrahymena group I intron, as well as tRNA(Phe) and tRNA(Asp) from yeast. Uranyl photocleavage was observed at specific positions in all molecules investigated. In order to characterize the sites, photocleavage was performed in the absence and in increasing amounts of MgCl(2). Uranyl photocleavage correlates well with sites of low calculated accessibility, suggesting that uranyl ions bind in tight RNA pockets formed by close approach of phosphate groups. RNA foldings require ion binding, usually magnesium ions. Thus, upon the adoption of the native structure, uranyl ions can no longer bind well except in flexible and open to the solvent regions that can undergo induced-fit without disrupting the native fold. Uranyl photocleavage was compared to N-ethyl-N-nitrosourea and lead-induced cleavages in the context of the three-dimensional X-ray structures. Overall, the regions protected from ENU attack are sites of uranyl cleavage, indicating sites of low accessibility which can form ion binding sites. On the contrary, lead cleavages occur at flexible and accessible sites and correlate with the unspecific cleavages prevalent in dynamic and open regions. Applied in a magnesium-dependent manner, and only in combination with other backbone probing agents such as N-ethyl-N-nitrosourea, lead and Fenton cleavage, uranyl probing has the potential to reveal high-affinity metal ion environments, as well as regions involved in conformational transitions.

Animals↗
PubMed1999

Complementarity between 3' terminal nucleotides of tRNA and primer binding site is a major determinant for selection of the tRNA primer used for initiation of HIV-1 reverse transcription.

The initiation of reverse transcription of human immunodeficiency virus type 1 (HIV-1) exclusively utilizes tRNALys,3 as a primer. Previous studies have shown that HIV-1 could use alternative tRNAs, such as tRNAIle or tRNAHis, to initiate reverse transcription only if the primer binding site (PBS) was made complementary to the 3' terminal 18 nucleotides of the cognate tRNA. However, upon in vitro culture, the viruses with a PBS complementary to the alternative tRNAs rapidly reverted to generate a PBS complementary to tRNALys,3. To investigate the process of reversion, we have constructed defective proviral genomes that contain a PBS complementary to tRNAIle or tRNAHis. The genomes contain the gene for xanthine-guanosine phosphoribosyl transferase (gpt) in place of env. Cotransfection of these proviral genomes with a plasmid-encoding vesicular stomatitis virus G protein (VSV-G) results in viruses that undergo a single round of HIV-1 infection; successful infections are scored as cells resistant to the drug mycophenolic acid. Using this single-round infection system, we demonstrated that HIV-1 with a PBS complementary to tRNAIle or tRNAHis is three- to fivefold less efficient in replication as measured by production of drug-resistant cell colonies compared to the wild-type virus. These viruses predominantly used the cognate tRNA as primer in their initial round of replication, although we did obtain a single cell colony in which the PBS was complementary to tRNALys,3. Using an HIV-1 provirus with a PBS complementary to yeast tRNAPhe, we established a single-round infection system in which the infectivity of this mutant HIV-1 relies on transfected yeast tRNAPhe. The results of our studies suggest that the mechanism for selection of the tRNA primer for initiation of reverse transcription relies primarily on the complementarity between the tRNA primerthe PBS.

Binding Sites↗
PubMed1990

Role of the tertiary nucleotides in the interaction of yeast phenylalanine tRNA with its cognate synthetase.

In vitro transcription by T7 RNA polymerase was used to prepare 32 different mutations in the 21 nucleotides that participate in the 9 tertiary base pairs or triples of yeast tRNAPhe. The mutations were designed either to disrupt the tertiary interaction or to change the sequence without disrupting the structure by transplanting tertiary interactions present in other tRNAs. Steady-state aminoacylation kinetics with purified yeast phenylalanyl synthetase revealed little change in reaction rate as long as a tertiary interaction was maintained. This suggests that the tertiary nucleotides only contribute to the folding of tRNAPhe and do not participate directly in sequence-specific interaction with the synthetase.

Amino Acyl-tRNA Synthetases↗
PubMed1998

Selective recognition and cleavage of RNA loop structures by Ni(II).Xaa-Gly-His metallopeptides.

The recognition and cleavage of tRNAPhe and the TAR RNA of HIV-1 by metallopeptides of the general form Ni(II).Xaa-Gly-His (where Xaa is Gly, Lys, or Arg) were investigated. The results of RNA cleavage analyses suggest that KHSO5- or magnesium monoperoxyphthalate-activated metallopeptides (1) induce nucleobase damage which requires aniline acetate for complete RNA strand scission and (2) selectively target the loops of stem-loop structures of the above-named substrates. In targeting RNA loop regions, the metallopeptides may be sensitive to intraloop structural features, including the overall structural environment of the loop itself and possibly the presence of intraloop hydrogen bonding. Overall, these results suggest that the metallopeptides interact selectively within a loop, in a fashion reminiscent of many RNA binding proteins, instead of targeting RNA single-stranded character alone. These observations further suggest a possible metallopeptide-based strategy for the molecular recognition of native RNA structures and insight with regard to the general features available for ligand binding site discrimination.

Base Sequence↗
PubMed2004

New class of bacterial phenylalanyl-tRNA synthetase inhibitors with high potency and broad-spectrum activity.

Phenylalanyl (Phe)-tRNA synthetase (Phe-RS) is an essential enzyme which catalyzes the transfer of phenylalanine to the Phe-specific transfer RNA (tRNA(Phe)), a key step in protein biosynthesis. Phenyl-thiazolylurea-sulfonamides were identified as a novel class of potent inhibitors of bacterial Phe-RS by high-throughput screening and chemical variation of the screening hit. The compounds inhibit Phe-RS of Escherichia coli, Haemophilus influenzae, Streptococcus pneumoniae, and Staphylococcus aureus, with 50% inhibitory concentrations in the nanomolar range. Enzyme kinetic measurements demonstrated that the compounds bind competitively with respect to the natural substrate Phe. All derivatives are highly selective for the bacterial Phe-RS versus the corresponding mammalian cytoplasmic and human mitochondrial enzymes. Phenyl-thiazolylurea-sulfonamides displayed good in vitro activity against Staphylococcus, Streptococcus, Haemophilus, and Moraxella strains, reaching MICs below 1 micro g/ml. The antibacterial activity was partly antagonized by increasing concentrations of Phe in the culture broth in accordance with the competitive binding mode. Further evidence that inhibition of tRNA(Phe) charging is the antibacterial principle of this compound class was obtained by proteome analysis of Bacillus subtilis. Here, the phenyl-thiazolylurea-sulfonamides induced a protein pattern indicative of the stringent response. In addition, an E. coli strain carrying a relA mutation and defective in stringent response was more susceptible than its isogenic relA(+) parent strain. In vivo efficacy was investigated in a murine S. aureus sepsis model and a S. pneumoniae sepsis model in rats. Treatment with the phenyl-thiazolylurea-sulfonamides reduced the bacterial titer in various organs by up to 3 log units, supporting the potential value of Phe-RS as a target in antibacterial therapy.

Animals↗
PubMed1998

Minimal template requirements for initiation of minus-strand synthesis in vitro by the RNA-dependent RNA polymerase of turnip yellow mosaic virus.

From mutational analysis of the 3'-terminal hairpin of turnip yellow mosaic virus (TYMV) RNA and use of nonstructured C-rich RNA templates, we conclude that the main determinant in the tRNA-like structure of TYMV RNA for initiation of minus-strand synthesis by the viral RNA-dependent RNA polymerase (RdRp) is the non-base-paired 3' ACC(A) end. Base pairing of this 3' end reduces the transcription efficiency drastically, and deletion of only the 3'-terminal A residue results in a fivefold drop in efficiency. The two C residues of the 3' ACCA end are required for efficient transcription, as shown by substitution mutations. However, the 5' A residue is not specifically involved in initiation of transcription, as shown by substitution mutations. Furthermore, the hairpin stem and loop upstream of the 3' ACCA end also do not interact with the RdRp in a base-specific way. However, for efficient transcription, the hairpin stem should be at least five bp in length, while the calculated deltaG value should be less than -10.5 kcal/mol. Unexpectedly, the use of nonstructured C-rich RNA templates showed that the RdRp can start internally on an NCCN or NUCN sequence. Therefore, a possible function of the tRNA-like structure of TYMV RNA may be to prevent internal initiation of minus-strand synthesis.

Base Sequence↗
PubMed1990

Recognition of the tRNA-like structure in tobacco mosaic viral RNA by ATP/CTP:tRNA nucleotidyltransferases from Escherichia coli and Saccharomyces cerevisiae.

The 3'-terminal tRNA-like structure of the tobacco mosaic virus RNA interacts with ATP/CTP:tRNA nucleotidyltransferases from Escherichia coli or yeast in much the same manner as do tRNAs. Primary sites of interaction cluster near the 3' end and in the loop proposed to be analogous to the psi-loop of a tRNA. Some modified bases in the tRNA-like structure inhibit interaction with nucleotidyltransferase, yet the analogous bases in a tRNA do not. The location of some of these nucleotides within the analog to the psi-loop suggests that this structure differs slightly from its counterpart in a tRNA. The location of other such bases in the helical stem near the 3' end can be explained if the pseudoknot is disrupted by these modified bases or if the tertiary structure of the RNA is altered in the enzyme-RNA complex. A partially denatured secondary structure that persists on denaturing gels is proposed.

Base Sequence↗
PubMed1984

Immunoelectron microscopic localization of the S19 site on the 30 S ribosomal subunit which is crosslinked to A site bound transfer RNA.

Phe-tRNA of Escherichia coli, specifically derivatized at the S4U8 position with the 9 A long p-azidophenacyl photoaffinity probe, was crosslinked exclusively to protein S19 of the 30 S ribosomal subunit when the transfer RNA occupied the ribosomal A site (Lin et al., 1983). Two antigenic sites for S19 are known, on opposite sides of the head of the subunit. In this work, discrimination between these two sites was accomplished by affinity immunoelectron microscopy. A dinitrophenyl group was placed on the acp3U47 residue of the same tRNA molecules bearing the photoprobe on S4U8. Addition of this group affected neither aminoacylation, A site binding, nor crosslinking. It also made possible specific affinity purification of crosslinked tRNA-30 S complexes from unreactive 30 S. Reaction of the 2,4-dinitrophenyl-labeled tRNA-30 S complex with antibody was followed by immunoelectron microscopy to reveal the sites of attachment. All of the bound antibody was associated with the ribosome region corresponding to only one of the two known antigenic sites for S19, namely the one closer to the large side projection of the 30 S subunit. A site within this region must be within 10 A of the S4U8 residue of tRNA when it is bound in the ribosomal A site.

Azides↗
PubMed1974

Correlation between three-dimensional structure and chemical reactivity of transfer RNA.

The bases of yeast tRNA(Phe) which react with carbodiimide and methoxyamine have been determined and this information has been combined with chemical modification studies of other workers to produce a composite picture of base accessibility in this tRNA. The results are compared with the three-dimensional structure which we have recently determined. The bases which react chemically lie in exposed positions in the three-dimensional model and those which do not are either in the double helical stem regions or else are involved in maintaining the tertiary structure through pairing or stacking interactions.

Base Sequence↗
PubMed2003

A peptide from the extension of Lys-tRNA synthetase binds to transfer RNA and DNA.

Eukaryotic aminoacyl-tRNA synthetases have dispensable extensions appended at the amino- or carboxyl-terminus as compared to their bacterial counterparts. While a synthetic peptide corresponding to the basic amino-terminal extension in yeast Asp-tRNA synthetase binds to DNA, the extension in the intact protein evidently binds to tRNA and enhances the tRNA specificity of Asp-tRNA synthetase. On the other hand, the amino-terminal extension in human Asp-tRNA synthetase, both within the intact protein and as a synthetic peptide, binds to tRNA. Here, the tRNA binding of a synthetic peptide, hKRS(Arg(25)-Glu(42)), corresponding to the amino-terminal extension of human Lys-tRNA synthetase (hKRS) was analyzed. This basic peptide bound to tRNA(Phe) and the apparent-binding constant increased with increasing concentrations of Mg(2+). The hKRS peptide also bound to DNA and polyphosphate; however, the apparent DNA-binding constants decreased at increasing concentrations of Mg(2+). The ability of the hKRS peptide to adopt alpha-helical conformation was demonstrated by NMR and circular dichroism. A Lys-rich peptide derived from the elongation factor 1alpha was also examined and bound to DNA but not to tRNA.

Amino Acid Sequence↗