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At least 19 recordsLinked to original sources
PubMed1992

Temperature regulation of Shigella virulence: identification of the repressor gene virR, an analogue of hns, and partial complementation by tyrosyl transfer RNA (tRNA1(Tyr)).

virR is the central regulatory locus required for coordinate temperature-regulated virulence gene expression in the human enteric pathogens of Shigella species. Detailed characterization of VirR+ clones revealed that virR consisted of a 411 bp open reading frame (ORF) that mapped to a chromosomally located 1.8kb EcoRI-AccI DNA fragment from Shigella flexneri. Insertional inactivation of the virR ORF at a unique HpaI restriction site resulted in a loss of VirR+ activity. The virR ORF nucleotide sequence was virtually identical to the Escherichia coli hns gene, which encodes the histone-like protein, H-NS. Based on the predicted amino acid sequence of E. coli H-NS, only a single conservative base-pair change was identified in the virR gene. An additional clone, designated VirRP, which only partially complemented the virR mutation, was also characterized and determined by Southern hybridization and nucleotide sequence analysis to be unique from virR. Subclone mapping of this clone indicated that the VirRP phenotype was a result of the multiple copy expression of the S. flexneri gene for tRNA(Tyr). These data constitute the first direct genetic evidence that virR is an analogue of the E. coli hns gene, and suggest a model for temperature regulation of Shigella species virulence via the bacterial translational machinery.

Bacterial Outer Membrane Proteins↗
PubMed1991

Identity determinants of E. coli tRNAs.

Identity determinants of E. coli tRNA(Val) and three class II tRNAs, tRNA(Ser), tRNA(Tyr) and tRNA(Leu), are studied by using various variants of tRNA transcripts. Anticodon, discriminator base and acceptor stem are involved in the identity elements for tRNA(Val). Discrimination among class II tRNAs are considered to be dependent on the bases at positions 2, 71 and 73 as well as their different tertiary structures including the long variable arm.

Codon↗
PubMed1998

Transfer RNA identity rules and conformation of the tyrosine tRNA-like domain of BMV RNA imply additional charging by histidine and valine.

This paper reports the first example of a triple aminoacylation specificity of a viral tRNA-like domain. These findings were based on structural studies on the brome mosaic virus (BMV) tRNA-like domain (Felden et al., 1994, J. Mol. Biol. 235, 508-531) together with knowledge on tRNA aminoacylation identity rules suggesting potential histidinylation and valylation capacities of the viral RNA in addition to its already known tyrosylation ability. Here, both predictions are demonstrated by in vitro aminoacylation assays. Kinetic parameters of histidinylation and valylation of BMV tRNA-like structure have been determined and compared to those of the corresponding tRNA transcripts and to the tyrosylation capacity of the molecule. The influence of experimental conditions on aminoacylation reactions was also studied. The novel aminoacylation capacities of BMV tRNA-like domain support its already reported three-dimensional fold and illustrate the predictive potential of modeling data. Biological necessity of specific or non specific aminoacylation will be discussed.

Base Sequence↗
PubMed1996

Maturation pathways for E. coli tRNA precursors: a random multienzyme process in vivo.

tRNA maturation consists of the specific removal of precursor sequences from both the 5' and 3' termini of an initial RNA transcript. How this is accomplished has heretofore not been ascertained in any system. Using Northern analysis of RNA isolated from a variety of RNase-deficient E. coli strains, we have identified the processing intermediates that accumulate in the absence of specific processing nucleases. From this information we have established the maturation pathways for 12 different E. coli tRNAs including the specific role of each of the relevant RNases in the process. The surprising conclusion from this work is that tRNA maturation is a stochastic process that lacks a defined order and that can proceed with a variety of alternative 3' processing nucleases.

Base Sequence↗
PubMed1981

The genes coding for tRNA Tyr of Drosophila melanogaster: localization of determination of the gene numbers.

Transfer RNA(Tyr) (anticodon G psi A) was isolated from Drosophila melanogaster by means of Sepharose 4B, RPC-5, and polyacrylamide gel electrophoresis. The rRNA was iodinated in vitro with Na125 I and hybridized in situ to salivary gland chromosomes from Drosophila. The genes of rRNA(Tyr) were localized in eight regions of the genome by autoradiography. Restriction enzyme analysis of genomic DNA indicated that the haploid Drosophila genome codes for about 23 tRNA(Tyr) genes. The regions 22F and 85A each contain four to five tRNA(Tyr) genes, whereas the regions 28C, 41AB, 42A, 42E, and 56D each contain two to three tRNA(Tyr) genes.

Animals↗
PubMed1995

Macromolecular recognition through electrostatic repulsion.

In the process of genetic translation, each aminoacyl-tRNA synthetase specifically aminoacylates its cognate tRNAs and rejects the 19 other species of tRNAs. A decrease in the specificity of this reaction can result in misincorporations of amino acids into proteins and be deleterious to the cell. In the case of tyrosyl-tRNA synthetase from Bacillus stearothermophilus, the change of residue Glu152 into Ala results in erroneous interactions with non-cognate tRNAs. To analyse how Glu152 contributes to the discrimination between tRNAs by tyrosyl-tRNA synthetase, 11 changes to this residue were created by mutagenesis. The misaminoacylations of tRNA(Phe) and tRNA(Val) with tyrosine in vitro (on a scale going from 1 to 30) and the toxicity of tyrosyl-tRNA synthetase in vivo (on a scale from 1 to 10(7)) increased in a correlated way when the nature of the side chain in position 152 varied from negatively charged to uncharged then to positively charged. The aminoacylation of tRNA(Tyr) was unaffected by the mutations. The results show that the role of Glu152 in the discrimination between tRNAs is purely negative, that it acts by electrostatic repulsion of non-cognate tRNAs and that this mechanism has been conserved throughout evolution.

Biological Evolution↗
PubMed1995

Ty3 transposes in mating populations of yeast: a novel transposition assay for Ty3.

Ty3 is a retrotransposon of Saccharomyces cerevisiae that integrates just upstream of the transcription initiation site of genes transcribed by RNA polymerase III. Ty3 transcription is pheromone-inducible in haploid cells and is mating-type regulated in diploid cells. The specificity of Ty3 integration was exploited in the design of a novel target into which transposition of Ty3 elements could be selected. The target plasmid contains divergently oriented tRNA genes with 19 base pairs separating the two tRNA gene coding sequences. An inactive ochre suppressor tRNA(Tyr) gene with a modified transcription initiation region was used as the selectable marker and a tRNA(Val) (AAC) gene was used to direct Ty3 integration into the transcription initiation region of the suppressor tRNA(Tyr) gene. Integration of Ty3 activated expression of the suppressor tRNA gene, which resulted in suppression of ochre nonsense alleles ade2-101(0) and lys2-1(0) and allowed cell growth on selective medium. Based on the activity of this target, Ty3, under control of a galactose-inducible promoter and present on a high copy-number plasmid, was estimated to transpose into the genome at a rate of 5.6 x 10(-3) per cell division. We show here that induction of Ty3 transcription from its natural promoter results in transposition. Ty3 elements in strains of the a or alpha mating-type transposed efficiently to target plasmids in cells of the opposite mating-type. Thus, natural transposition of Ty3 is regulated temporally to occur in mating populations.

Adenine↗
PubMed1994

Solution structure of the 3'-end of brome mosaic virus genomic RNAs. Conformational mimicry with canonical tRNAs.

The conformation of the last 201 nucleotides located at the 3'-end of brome mosaic virus (BMV) RNAs was investigated in solution using different chemical and enzymatic probes. Bases were probed with dimethylsulfate (which methylates N-1 positions of A, N-3 positions of C and N-7 positions of G), a carbodiimide (which modifies N-1 positions of G and N-3 positions of U) and diethylpyrocarbonate (which modifies N-7 positions of A). Ribonucleases T1, U2 and S1 were used to map unpaired nucleotides and ribonuclease V1 to monitor paired bases or stacked nucleotides. Cleavage or modification sites were detected by gel electrophoresis either indirectly by analyzing DNA sequence patterns generated by primer extension with reverse transcriptase of the modified RNAs or by direct identification within the statistical cleavage patterns of the RNA. On the basis of these biochemical results, an atomic model was built by computer modeling and its stereochemistry refined. The deduced secondary structure of the RNA confirms data previously proposed by others but contains additional base-pairs (A27-U32, A28-G31, G41-A134, G64-C68, U80-A99, G81-A98, G88-U91, G100-U126, U104-U125, G162-G166 and A172-A191), one new tertiary long-range interaction (U103-U164) and a small triple helical conformation with (G41-A134)-A18 and (C42-G133)-A17 interactions. The new secondary structure also indicates the existence of a second pseudoknot involving pairing between residues A181 to A184 and residues U197 to U194, outside the domain conferring tyrosylation ability to BMV RNA. The main outcome from the model stems from its intricate folding, which allows a new assignment for the domains mimicking the anticodon- and D-loop regions of tRNA. Interestingly, the stem and loop region found structurally to be analogous to the anticodon arm of tRNA(Tyr) does not contain the tyrosine anticodon involved in the aminoacylation process. The structural analogies with canonical tRNA(Tyr) illustrate the functional mimicry existing between the BMV RNA structure and canonical tRNA(Tyr) that allows for their efficient aminoacylation by tyrosyl-tRNA synthetase. This structural model rationalizes mutagenic and footprinting data that have established the importance of specific regions of the viral RNA for recognition by its replicase, (ATP,CTP):tRNA nucleotidyl-transferase and yeast tyrosyl-tRNA synthetase. The new fold has biological implications that can be used as a predictive tool for elaborating new experiments.

Base Sequence↗
PubMed1995

Requirement of RNA polymerase III transcription factors for in vitro position-specific integration of a retroviruslike element.

The yeast retroviruslike element Ty3 inserts at the transcription initiation sites of genes transcribed by RNA polymerase III (Pol III). An in vitro integration assay was developed with the use of Ty3 viruslike particles and a modified SUP2 tyrosine transfer RNA (tRNA(Tyr)) gene target. Integration was position-specific and required Ty3 integrase, Pol III transcription factor (TF) IIIB-, TFIIIC-, and Pol III-containing fractions showed that TFIIIB and TFIIIC, together, were sufficient for position-specific Ty3 integration, but not for transcription. This report demonstrates that in vitro integration of a retroelement can be targeted by cellular proteins.

DNA Nucleotidyltransferases↗
PubMed2001

Specific tyrosylation of the bulky tRNA-like structure of brome mosaic virus RNA relies solely on identity nucleotides present in its amino acid-accepting domain.

Residues specifying aminoacylation by yeast tyrosyl-tRNA synthetase (TyrRS) of the tRNA-like structure present at the 3'-end of brome mosaic virus (BMV) RNA were determined by the in vitro approach using phage T7 transcripts. They correspond to nucleotides equivalent to base-pair C1-G72 and discriminator base A73 in the amino acid-acceptor branch of the molecule. No functional equivalents of the tyrosine anticodon residues, shown to be weakly involved in tyrosine identity of canonical tRNA(Tyr), were found in the BMV tRNA-like structure. This indicates a behaviour of this large and intricate molecule reminiscent of that of a minihelix derived from an amino acid-acceptor branch. Furthermore, iodine footprinting experiments performed on a tyrosylable BMV RNA transcript of 196 nt complexed to yeast TyrRS indicate that the amino acid-acceptor branch of the viral RNA is protected against cleavages as well as a hairpin domain, which is possibly located perpendicularly to its accepting branch. This domain without the canonical anticodon loop or the tyrosine anticodon acts as an anchor for TyrRS interaction leading to a better efficiency of tyrosylation.

Acylation↗
PubMed1989

Structural basis for misaminoacylation by mutant E. coli glutaminyl-tRNA synthetase enzymes.

A single-site mutant of Escherichia coli glutaminyl-synthetase (D235N, GlnRS7) that incorrectly acylates in vivo the amber suppressor supF tyrosine transfer RNA (tRNA(Tyr] with glutamine has been described. Two additional mutant forms of the enzyme showing this misacylation property have now been isolated in vivo (D235G, GlnRS10; I129T, GlnRS15). All three mischarging mutant enzymes still retain a certain degree of tRNA specificity; in vivo they acylate supE glutaminyl tRNA (tRNA(Gln] and supF tRNA(Tyr) but not a number of other suppressor tRNA's. These genetic experiments define two positions in GlnRS where amino acid substitution results in a relaxed specificity of tRNA discrimination. The crystal structure of the GlnRS:tRNA(Gln) complex provides a structural basis for interpreting these data. In the wild-type enzyme Asp235 makes sequence-specific hydrogen bonds through its side chain carboxylate group with base pair G3.C70 in the minor groove of the acceptor stem of the tRNA. This observation implicates base pair 3.70 as one of the identity determinants of tRNA(Gln). Isoleucine 129 is positioned adjacent to the phosphate of nucleotide C74, which forms part of a hairpin structure adopted by the acceptor end of the complexed tRNA molecule. These results identify specific areas in the structure of the complex that are critical to accurate tRNA discrimination by GlnRS.

Acylation↗
PubMed1974

Effects of guanosine tetraphosphate, guanosine pentaphosphate, and beta-gamma methylenyl-guanosine pentaphosphate on gene expression of Escherichia coli in vitro.

The effects of guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp), both produced by E. coli, were measured on the activities of several genes in a cell-free system. Gene activity is measured as gene-directed synthesis of biochemically competent protein or transfer RNA. Both ppGpp and pppGpp stimulated the activities of the ara, lac, and trp operons and inhibited the arg operon. Production of transfer-RNA(Tyr) was unaffected by moderate levels of either ppGpp or pppGpp and only slightly inhibited at higher levels of ppGpp. Since the cell-free reaction mixtures hydrolyze pppGpp to ppGpp, we performed similar studies with a hydrolysis-resistant analog of pppGpp, the beta-gamma methylenyl derivative (pcppGpp). In general, pcppGpp shows the same inhibitory potency as pppGpp for the arg operon, but lacks the stimulatory effects on the ara, lac, and trp operons. This result suggests that the stimulation of these gene activities is specific for ppGpp.Under similar conditions, pppGpp and ppGpp show a slight inhibitory effect on the messenger-directed synthesis of beta-galactosidase and no effect on the messenger-directed synthesis of MS2 viral-coat protein. These observations, together with the fact that in the same system these nucleotides affect coupled transcription and translation, lead us to surmise that the activities of pppGpp and ppGpp are exerted at the level of RNA polymerase activity.

Acetamides↗
PubMed1988

Characterization of phage-specific transfer RNA molecules coded by Vibrio eltor phage e4.

Transfer RNAs were isolated from phage e4-infected Vibrio eltor Mak 757 cells. These were aminoacylated with 14 individual 3H-labeled L-amino acids. Hybridization of these [3H]aminoacyl-tRNAs with phage e4 DNA revealed that the phage e4 encodes tRNAs for arginine, tryptophan, tyrosine, leucine, and isoleucine. Direct aminoacylation of phage-coded tRNA molecules isolated from phage DNA-RNA hybrids also confirmed this observation.

Bacteriophages↗
PubMed1999

Targeted cleavage of RNA molecules by human RNase P using minimized external guide sequences.

The endoribonuclease RNase P processes tRNA-like structures that are assembled out of two separate strands. In these bimolecular constructs, one of the strands is cleaved by the enzyme, and the other one is called the external guide sequence (EGS). A number of EGS with different mutations and deletions were tested for the ability to induce cleavage with human RNase P. Different domains of the original tRNAtyr-like structure were deleted or modified. The anticodon stem and loop and the variable loop could be deleted without a detrimental effect on recognition by RNase P. Modifications in the lengths of T stem and aminoacyl acceptor stem led to a decrease in the relative amount of cleavage, whereas modifications of the D stem were more permissible. Single nucleotide deletions in the T loop reduced cleavage to different extents, depending on the position. Values for the Kd of complex formation of bimolecular constructs with annealing arms of varying lengths ranged from 0.2 nM to 28 nM. A cleavage rate of 1 min(-1) was measured for both the bimolecular target-EGS complex and tRNA precursor.

Anticodon↗
PubMed1989

Structural analogies between the 3' tRNA-like structure of brome mosaic virus RNA and yeast tRNATyr revealed by protection studies with yeast tyrosyl-tRNA synthetase.

Contacts between the tRNA-like domain in brome mosaic virus RNA and yeast tyrosyl-tRNA synthetase have been determined by footprinting with enzymatic probes. Regions in which the synthetase caused protections indicative of direct interaction coincide with loci identified by mutational studies as being important for efficient tyrosylation [Dreher, T. W. & Hall, T. C. (1988) J. Mol. Biol. 201, 41-55]. Additional extensive contacts were found upstream of the core of the tRNA-like structure. In parallel, the contacts of yeast tRNATyr with its cognate synthetase were determined by the same methodology and comparison of protected nucleotides in the two RNAs has permitted the assignment of structural analogies between domains in the viral tRNA-like structure and tRNATyr. Amino acid acceptor stems are similarly recognized by yeast tyrosyl-tRNA synthetase in the two RNAs, indicating that the pseudoknotted fold in the viral RNA does not perturb the interaction with the synthetase. A further important analogy appears between the anticodon/D arm of the L-conformation of tRNAs and a complex branched arm of the viral tRNA-like structure. However, no apparent anticodon triplet exists in the viral RNA. These results suggest that the major determinants for tyrosylation of yeast tRNATyr lie outside the anticodon stem and loop, possibly in the amino acid acceptor stem.

Acylation↗
PubMed2002

Role of an essential triloop hairpin and flanking structures in the 3' untranslated region of Alfalfa mosaic virus RNA in in vitro transcription.

The minus-strand promoter of Alfalfa mosaic virus (AMV), a tripartite plant virus belonging to the family Bromoviridae, is located within the 3'-terminal 145 nucleotides (nt), which can adopt a tRNA-like structure (TLS). This contrasts with the subgenomic promoter for RNA4 synthesis, which requires approximately 40 nt and forms a single triloop hairpin. Detailed analysis of the minus-strand promoter now shows that a similar triloop hairpin, hairpin E (hpE), is crucial for minus-strand synthesis. The loop sequence of hpE appeared to not be essential for RNA synthesis, whereas the identity and base-pairing capability of bases below the triloop were indeed essential. Reducing the size of the bulge loop of hpE triggered transcription from an internal site similar to the process of subgenomic transcription. Similar effects were observed when deleting (part of) the TLS, suggesting that tertiary contacts between hpE and the TLS prevent internal initiation. The data indicate that the minus-strand promoter hpE and the subgenomic promoter hairpin are equivalent in binding the viral polymerase. We propose that the major role of the TLS is to enforce the initiation of transcription by polymerase at the very 3' end of the genome.

3' Untranslated Regions↗
PubMed2001

Interaction of the Neurospora crassa mitochondrial tyrosyl-tRNA synthetase (CYT-18 protein) with the group I intron P4-P6 domain. Thermodynamic analysis and the role of metal ions.

The Neurospora crassa mitochondrial tyrosyl-tRNA synthetase (CYT-18 protein) functions in splicing group I introns by promoting the formation of the catalytically active structure of the intron's catalytic core. Previous studies suggested a model in which the protein binds first to the intron's P4-P6 domain, and then makes additional contacts with the P3-P9 domain to stabilize the two domains in the correct relative orientation to form the intron's active site. Here, we analyzed the interaction of CYT-18 with a small RNA (P4-P6 RNA) corresponding to the isolated P4-P6 domain of the N. crassa mitochondrial large subunit ribosomal RNA intron. RNA footprinting and modification-interference experiments showed that CYT-18 binds to this small RNA around the junction of the P4-P6 stacked helices on the side opposite the active-site cleft, as it does to the P4-P6 domain in the intact intron. The binding is inhibited by chemical modifications that disrupt base-pairing in P4, P6, and P6a, indicating that a partially folded structure of the P4-P6 domain is required. The temperature-dependence of binding indicates that the interaction is driven by a favorable enthalpy change, but is accompanied by an unfavorable entropy change. The latter may reflect entropically unfavorable conformational changes or decreased conformational flexibility in the complex. CYT-18 binding is inhibited at > or =125 mM KCl, indicating a strong dependence on phosphodiester-backbone interactions. On the other hand, Mg(2+) is absolutely required for CYT-18 binding, with titration experiments showing approximately 1.5 magnesium ions bound per complex. Metal ion-cleavage experiments identified a divalent cation-binding site near the boundary of P6 and J6/6a, and chemical modification showed that Mg(2+) binding induces RNA conformational changes in this region, as well as elsewhere, particularly in J4/5. Together, these findings suggest a model in which the binding of Mg(2+) near J6/6a and possibly at one additional location in the P4-P6 RNA induces formation of a specific phosphodiester-backbone geometry that is required for CYT-18 binding. The binding of CYT-18 may then establish the correct structure at the junction of the P4/P6 stacked helices for assembly of the P3-P9 domain. The interaction of CYT-18 with the P4-P6 domain appears similar to the TyrRS interaction with the D-/anticodon arm stacked helices of tRNA(Tyr).

Base Sequence↗