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PubMed2002

Three modifications in the D and T arms of tRNA influence translation in Escherichia coli and expression of virulence genes in Shigella flexneri.

The modified nucleosides 2'-O-methylguanosine, present at position 18 (Gm18), 5-methyluridine, present at position 54 (m(5)U54), and pseudouridine, present at position 55 (Psi55), are located in the D and T arms of tRNAs and are close in space in the three-dimensional (3D) structure of this molecule in the bacterium Escherichia coli. The formation of these modified nucleosides is catalyzed by the products of genes trmH (Gm18), trmA (m(5)U54), and truB (Psi55). The combination of trmH, trmA, and truB mutations resulting in lack of these three modifications reduced the growth rate, especially at high temperature. Moreover, the lack of three modified nucleotides in tRNA induced defects in the translation of certain codons, sensitivity to amino acid analog 3,4-dehydro-DL-proline, and an altered oxidation of some carbon compounds. The results are consistent with the suggestion that these modified nucleosides, two of which directly interact in the 3D structure of tRNA by forming a hydrogen bond between Psi55 and Gm18, stabilize the structure of the tRNA. Moreover, lack of Psi55 in tRNA of human pathogen Shigella flexneri leads to a reduced expression of several virulence-associated genes.

Bacterial Proteins↗
PubMedDate not supplied

[Interaction of oligonucleotides and ATP with preparations of sIgA possessing protein kinase activity].

Interaction of secretory immunoglobulins A of a varying degree of purity with oligonucleotides and ATP has been studied by the method of affinity modification. For this aim we used reactive derivatives of 32P-labeled deoxyoligonucleotide (ClRCH2NHp(T)14) and [gamma-32P]ATP (ClR-32PppA) or ATP (ClR-pppA) bearing a 4-[(N-2-chloroethyl-N-methyl)amino]benzylamine residue. Preparations of sIgA were obtained from human milk by sequential chromatography on protein A-sepharose (P1), DEAE-fractogel (P2) and by gel-filtration in 50 mM NaOH (P3). It was revealed, that the H- and L-chains of sIgA P1; H-, L-chains and secretory component (SC) in sIgA P2 and only SC in sIgA P3 were exposed to modification after incubation with ClRCH2NHp(T)14. LPS, DNA, tRNA, heparin, sufficiently inhibited the modification of chains of sIgA P1. These competitors did not influence the modification of H- and L-chains of sIgA P2, but DNA, tRNA, heparin, inhibited binding of SC with the modifier. Suppressing affect of binding of ClRCH2NHp(T)14 with secretory component of sIgA P3 by d(T)14 has been observed as well. The research of ClR-32PppA interaction with sIgA P3 has shown that H- and L-chains of sIgA are exposed to modification. ATP inhibited the reaction. Study of the influence of modification on the protein kinase activity of sIgA P3 has revealed, that the preliminary incubation of sIgA P3 with ClR-pppA leads to inhibition of protein kinase activity. We suggest that sIgA, possessing the protein kinase activity (sIgA-abzymes) has an ATP-binding center (catalytic center) and has an oligonucleotide-binding center as well.

Adenosine Triphosphate↗
PubMed1998

Codon usages in different gene classes of the Escherichia coli genome.

A new measure for assessing codon bias of one group of genes with respect to a second group of genes is introduced. In this formulation, codon bias correlations for Escherichia coli genes are evaluated for level of expression, for contrasts along genes, for genes in different 200 kb (or longer) contigs around the genome, for effects of gene size, for variation over different function classes, for codon bias in relation to possible lateral transfer and for dicodon bias for some gene classes. Among the function classes, codon biases of ribosomal proteins are the most deviant from the codon frequencies of the average E. coli gene. Other classes of 'highly expressed genes' (e.g. amino acyl tRNA synthetases, chaperonins, modification genes essential to translation activities) show less extreme codon biases. Consistently for genes with experimentally determined expression rates in the exponential growth phase, those of highest molar abundances are more deviant from the average gene codon frequencies and are more similar in codon frequencies to the average ribosomal protein gene. Independent of gene size, the codon biases in the 5' third of genes deviate by more than a factor of two from those in the middle and 3' thirds. In this context, there appear to be conflicting selection pressures imposed by the constraints of ribosomal binding, or more generally the early phase of protein synthesis (about the first 50 codons) may be more biased than the complete nascent polypeptide. In partitioning the E. coli genome into 10 equal lengths, pronounced differences in codon site 3 G+C frequencies accumulate. Genes near to oriC have 5% greater codon site 3 G+C frequencies than do genes from the ter region. This difference also is observed between small (100-300 codons) and large (>800 codons) genes. This result contrasts with that for eukaryotic genomes (including human, Caenorhabditis elegans and yeast) where long genes tend to have site 3 more AT rich than short genes. Many of the above results are special for E. coli genes and do not apply to genes of most bacterial genomes. A gene is defined as alien (possibly horizontally transferred) if its codon bias relative to the average gene exceeds a high threshold and the codon bias relative to ribosomal proteins is also appropriately high. These are identified, including four clusters (operons). The bulk of these genes have no known function.

Amino Acyl-tRNA Synthetases↗
PubMedDate not supplied

[Study of the poly(U)-dependent interaction of tRNA(Phe) with the P-site of Escherichia coli ribosomes by chemical modification with nitrosoethylurea].

The accessibility of phosphates in E. coli tRNA(Phe) bound to E. coli ribosomes and 30S subunits for attack by an alkylating agent ethylnitrosourea was studied. The experimental technique allowed us to investigate a part of the molecule between N-11 and N-72. Being bound to the poly(U)-programmed P site of 30S subunit, tRNA(Phe) reveals protection in a discrete region including phosphates 23-44. This region corresponds to the hairpin formed from the anticodon arm and the adjoining 3' strand of the D stem and the variable loop. On the P site of 70S-poly(U), additional protection was observed in a region between N-45 and N-63. This region corresponds to the extra loop and T stem, and reveals a fine structure of protection: protected phosphates in positions 45-49, 51, 54-55, 58-61, and 63 alternate with unprotected ones in positions 50, 52-53, 56-57 and 62. We conclude that ethylnitrosourea can be used for detailed study of tRNA-ribosome contacts.

Anticodon↗
PubMedDate not supplied

Block-units method for conformational calculations of large nucleic acid chains. II. The two-hierarchical approach and its application to conformational arrangement of the unusual T psi C loop of rabbit tRNA(Val).

The two-level hierarchical methodology is suggested for conformational calculations of large fragments of nucleic acids. The method of the first level is intended for performing a fast screening of the conformational phase space. The high-level method may be used to refine structurally important conformations. The method of the first level is the block-units method, which has been developed specially for these purposes (see part I). It has been shown that the block-units method allows the satisfactory calculation of the structure parameters of the optimal conformations of polynucleotides. The results of the conformational rearrangement calculations of the T psi C loop of the tRNA(Phe) after modification of its sequence are represented.

Animals↗
PubMed1993

Identity determinants of human tRNA(Ser): sequence elements necessary for serylation and maturation of a tRNA with a long extra arm.

Recently, there has been much progress in understanding tRNA identity, i.e. in elucidating the sets of nucleotides that are responsible for the specific aminoacylation of a tRNA with its cognate amino acid. Interest focused, however, on tRNAs from Escherichia coli and yeast. Here we have identified the major and minor determinants of human tRNA(Ser) which were revealed by an identity switch from human tRNA(Val) to tRNA(Ser). We used in vitro transcripts and subsequent aminoacylation by HeLa S100 extract to determine the kinetic parameter Vmax/Km. The two major identity elements which are absolutely required for aminoacylation by human seryl-tRNA synthetase are the discriminator base and the long extra arm. This is in contrast to E. coli tRNA(Ser) where the discriminator base is unimportant, whereas identity determinants in the acceptor stem are required. Other sequence elements have an influence not only on serylation, but also on tRNA maturation in vitro, i.e. on pre-tRNA processing and base modification. These nucleotides are located in the DHU and the T phi C arm and are probably necessary for the proper folding of tRNAs containing a long extra arm. A34 to inosine modification depends highly on the correct three-dimensional structure of the tRNA, whereas A58 to m1A methylation does not rely on the three-dimensional folding of the substrate. This is the first tRNA identity switch involving the exchange of a short versus a long extra arm.

Acylation↗
PubMed1986

Mutation in the D arm enables a suppressor with a CUA anticodon to read both amber and ochre codons in Escherichia coli.

Su9 of Escherichia coli differs from tRNATrp by only a G to A transition in the D arm, yet has an enhanced ability to translate UGA by an unusual C X A wobble pairing. In order to examine the effects of this mutation on translation of the complementary and wobble codons in vivo, we constructed the gene for an amber (UAG) suppressing variant of Su9, trpT179, by making the additional nucleotide change required for an amber suppressor anticodon. The resultant suppressor tRNA, Su79, is a very strong amber suppressor. Furthermore, the D arm mutation enables Su79 to suppress ochre (UAA) codons by C X A wobble pairing. These data demonstrate that the effect of the D arm mutation on wobble pairing is not restricted to a CCA anticodon. The effect extends to the CUA anticodon of Su79, thereby creating a new type of ochre suppressor. The new coding activity of Su79 cannot be explained by alterations in the level of aminoacylation, steady-state tRNA concentration, or nucleotide modification. The A24 mutation could permit unorthodox wobble pairings by generally enhancing tRNA efficiency at all codons or by altering codon specificity.

Anticodon↗
PubMed2003

Structure of tRNA pseudouridine synthase TruB and its RNA complex: RNA recognition through a combination of rigid docking and induced fit.

RNA pseudouridine synthase, TruB, catalyzes pseudouridine formation at U55 in tRNA. This posttranscriptional modification is almost universally conserved and occurs in the T arm of most tRNAs. We determined the crystal structure of Escherichia coli TruB apo enzyme, as well as the structure of Thermotoga maritima TruB in complex with RNA. Comparison of the RNA-free and -bound forms of TruB reveals that this enzyme undergoes significant conformational changes on binding to its substrate. These conformational changes include the ordering of the "thumb loop," which binds right into the RNA hairpin loop, and a 10 degree hinge movement of the C-terminal domain. Along with the result of docking experiments performed on apo TruB, we conclude that TruB recognizes its RNA substrate through a combination of rigid docking and induced fit, with TruB first rigidly binding to its target and then maximizing the interaction by induced fit.

Binding Sites↗
PubMed1982

Nucleotide sequence of the SUF2 frameshift suppressor gene of Saccharomyces cerevisiae.

To elucidate the molecular mechanism of frameshift suppression by the SUF2 gene of yeast, the sequences of DNA fragments carrying the SUF2-1 and suf2+ alleles of the gene and surrounding regions have been determined. Comparison of the suppressor and wild-type sequences indicates that the SUF2 gene product is a proline tRNA. Disregarding possible base modifications, we find that the wild-type suf2+ anticodon of the tRNA inferred from the DNA sequence is 3'-GGA-5'. The SUF2-1 mutation represents the insertion of a G-C base pair at a position in the gene that corresponds to the anticodon loop of the tRNA. Replacement of the wild-type suf2+ anticodon by a 3'-GGGA-5' fourbase anticodon enables the SUF2-1 tRNA to suppress the 5'-CCCU-3' four-base codons generated as the result of the his4-712 and his4-713 frameshift mutations. This nontriplet codon-anticodon interaction restores the correct reading frame and allows synthesis of a functional his4 protein.

Base Sequence↗
PubMed1995

UV-A oxidative damage modified by environmental conditions in Escherichia coli.

The effect of sublethal fluences (50-200 kJ m-2) of UV-A radiation (320-400 nm) in bacterial cells is a transient growth inhibition related to photo-modified tRNA and is associated with changes in membrane structure and function. Higher UV-A fluences result in cell death due to the production of reactive oxygen species, so far undetected at sublethal doses. Oxidative mechanisms of toxicity induced by 120 kJ m-2 UV-A radiation can be recorded by ultra-weak chemiluminescence, useful in quantifying oxidative reactions. When Escherichia coli was exposed to UV-A stress at a fluence rate equivalent to that of the Sun in the biosphere (33 W m-2), chemiluminescence levels were proportional to the photodamage. Chemiluminescence and photo-damage are linearly proportional and dependent on environmental conditions of the cells. It is postulated that in addition to tRNA photo-modification, UV-A alters the membrane structure of E. coli by oxidative damage, since changes in the membrane structure under different environmental conditions play a key role in the cell's response to UV-A injury.

Cell Membrane↗
PubMed2005

Characterisation of the 11 Kb DNA region adjacent to the gene encoding Desulfovibrio gigas flavoredoxin.

Flavoredoxin is an FMN binding protein that functions as an electron carrier in the sulphate metabolism of Desulfovibrio gigas. The neighbouring DNA regions of the gene encoding flavoredoxin were sequenced and characterised. Transcript analysis of the flavoredoxin gene resulted in a positive band corresponding to the size of the coding region, suggesting that flavoredoxin is encoded by a monocystronic unit, as previously suggested by sequence analysis. Analysis of the adjacent DNA regions revealed several interesting genes. The sequenced DNA regions contain nine open reading frames (ORFs) organised in two polycystronic and two monocystronic units. These genes encode proteins involved in different metabolic pathways, namely in DNA methylation, tRNA and rRNA modification, mRNA metabolism, cell division, CoA synthesis and lipoprotein transport across the membrane.

Blotting, Northern↗
PubMed1982

The nucleotide sequence of the maize and spinach chloroplast isoleucine transfer RNA encoded in the 16S to 23S rDNA spacer.

The sequence of maize chloroplast tRNAIle2, encoded in the 16S to 23S rDNA spacer, was determined using in vitro labeling techniques. The sequence is: pG-G-G-C-U-A-U-U-A-G-C-U-C-A-G-U-Gm-G-D-A-G-A-G-C-m22G-C-G-C-C-C-C-U-G-A-U-t6A- A-G-G-G-C-G-A-G-m7G-acp3U-C-U-C-U-G-G-T-psi-C-A-A-G-U-C-C-A-G-G-A-U-G-G-C-C-C-A -C-C-AOH. This sequence is identical to that predicted from the corresponding gene sequence, after excision of a long intervening sequence (1), but shows the post-transcriptional modifications of this tRNA. Furthermore it demonstrates that the excision of the intron occurs after the second base following the anticodon and that this gene, which is over 1000 base-pair long, is transcribed and processed into a mature functional chloroplast-tRNA. The sequence of maize (a monocot) and spinach (a dicot) tRNAIle2 are shown to be identical.

Base Sequence↗
PubMed1989

Ribosomal proteins S7 and L1 are located close to the decoding site of E. coli ribosome--affinity labeling studies with modified tRNAs carrying photoreactive probes attached adjacent to the 3'-end of the anticodon.

Two photoreactive azidonitrophenyl probes have been attached to Yeast methionine elongator tRNA by chemical modification of the N6-(threoninocarbonyl)adenosine located next to the 3'-end of the anticodon. The maximum distance between the purine ring and the azido group estimated for the two probes is 16-17 and 23-24A, respectively. Binding and cross-linking of the uncharged, modified tRNAs to E. coli ribosomes have been studied with and without poly(A,U,G) as a message, under conditions directing uncharged tRNAs preferentially to the P-site. The modified tRNAs retain their binding activity and upon irradiation bind covalently to the ribosome with very high yields. Protein S7 is the major cross-linking target for both modified tRNAs, in the presence or absence of poly(A,U,G). Protein L1 and to a lesser extent proteins L33 and L27 have been found to be cross-linked with the short probe. Cross-linking to 168 rRNA reaches significant levels only in the absence of the message.

Affinity Labels↗
PubMed1996

Preparation of biologically active Ascaris suum mitochondrial tRNAMet with a TV-replacement loop by ligation of chemically synthesized RNA fragments.

Ascaris suum mitochondrial tRNA Met lacking the entire T stem was prepared by enzymatic ligation of two chemically synthesized RNA fragments. The synthetic tRNA could be charged with methionine by A.suum mitochondrial extract, although the charging activity was considerably low compared with that of the native tRNA, probably due to lack of modification. Enzymatic probing of the synthetic tRNA showed a very similar digestion pattern to that of the native tRNA Met, which has already been concluded to take an L-shape-like structure [Watanabe et al. (1994) J. Biol. Chem., 269, 22902-22906]. These results suggest that the synthetic tRNA possesses almost the same conformation as the native one, irrespective of the presence or absence of modified residues. The method of preparing the bizarre tRNA used here will provide a useful tool for elucidating the tertiary structure of such tRNAs, because they can be obtained without too much difficulty in the amounts necessary for physicochemical studies such as NMR spectroscopy.

Animals↗
PubMed2005

Detection and discovery of RNA modifications using microarrays.

Using a microarray that tiles all known yeast non-coding RNAs, we compared RNA from wild-type cells with RNA from mutants encoding known and putative RNA modifying enzymes. We show that at least five types of RNA modification (dihydrouridine, m1G, m2(2)G, m1A and m6(2)A) catalyzed by 10 different enzymes (Trm1p, Trm5, Trm10p, Dus1p-Dus4p, Dim1p, Gcd10p and Gcd14p) can be detected by virtue of differential hybridization to oligonucleotides on the array that are complementary to the modified sites. Using this approach, we identified a previously undetected m1A modification in GlnCTG tRNA, the formation of which is catalyzed by the Gcd10/Gcd14 complex. complex.

Mutation↗
PubMed2000

Posttranscriptional modification of transfer RNA in the submarine hyperthermophile Pyrolobus fumarii.

In the RNA of hyperthermophiles, which grow optimally between 80 degrees C and 106 degrees C, posttranscriptional modification has been identified as a leading mechanism of structural stabilization. Particularly in the Archaeal evolutionary domain these modifications are expressed as a structurally diverse array of modification motifs, many of which include ribose methylation. Using mass spectrometric techniques we have examined the posttranscriptional modifications in unfractionated tRNA from the remarkable organism Pyrolobus fumarii, which grows optimally at 106 degrees C, but up to 113 degrees C (Blöchl et al. (1997), Extremophiles, 1, 14-21). Twenty-six modified nucleosides were detected, 11 of which are methylated in ribose. A new RNA nucleoside, 1,2'-O-dimethylguanosine (m1Gm) was characterized and the structure confirmed by chemical synthesis.

Chromatography, High Pressure Liquid↗
PubMed2006

MCT-1 protein interacts with the cap complex and modulates messenger RNA translational profiles.

MCT-1 is an oncogene that was initially identified in a human T cell lymphoma and has been shown to induce cell proliferation as well as activate survival-related pathways. MCT-1 contains the PUA domain, a recently described RNA-binding domain that is found in several tRNA and rRNA modification enzymes. Here, we established that MCT-1 protein interacts with the cap complex through its PUA domain and recruits the density-regulated protein (DENR/DRP), containing the SUI1 translation initiation domain. Through the use of microarray analysis on polysome-associated mRNAs, we showed that up-regulation of MCT-1 was able to modulate the translation profiles of BCL2L2, TFDP1, MRE11A, cyclin D1, and E2F1 mRNAs, despite equivalent levels of mRNAs in the cytoplasm. Our data establish a role for MCT-1 in translational regulation, and support a linkage between translational control and oncogenesis.

Animals↗
PubMed1985

Temperature jump relaxation studies on the interactions between transfer RNAs with complementary anticodons. The effect of modified bases adjacent to the anticodon triplet.

We have used the temperature-jump relaxation technique to determine the kinetic and thermodynamic parameters for the association between the following tRNAs pairs having complementary anticodons: tRNA(Ser) with tRNA(Gly), tRNA(Cys) with tRNA(Ala) and tRNA(Trp) with tRNA(Pro). The anticodon sequence of E. coli tRNA(Ser), GGA, is complementary to the U*CC anticodon of E. coli tRNA(Gly(2] (where U* is a still unknown modified uridine base) and A37 is not modified in none of these two tRNAs. E. coli tRNA(Ala) has a VGC anticodon (V is 5-oxyacetic acid uridine) while tRNA(Cys) has the complementary GCA anticodon with a modified adenine on the 3' side, namely 2-methylthio N6-isopentenyl adenine (mS2i6A37) in E. Coli tRNA(Cys) and N6-isopentenyl adenine (i6A37) in yeast tRNA(Cys). The brewer yeast tRNA(Trp) (anticodon CmCA) differs from the wild type E. coli tRNA(Trp) (anticodon CCA) in several positions of the nucleotide sequence. Nevertheless, in the anticodon loop, only two interesting differences are present: A37 is not modified while C34 at the first anticodon position is modified into a ribose 2'-O methyl derivative (Cm). The corresponding complementary tRNA is E.coli tRNA(Pro) with the VGG anticodon. Our results indicate a dominant effect of the nature and sequence of the anticodon bases and their nearest neighbor in the anticodon loop (particularly at position 37 on the 3' side); no detectable influence of modifications in the other tRNA stems has been detected. We found a strong stabilizing effect of the methylthio group on i6A37 as compared to isopentenyl modification of the same residue. We have not been able so far to assess the effect of isopentenyl modification alone in comparison to unmodified A37. The results obtained with the complex yeast tRNA(Trp)-E.coli tRNA(Pro) also suggest that a modification of C34 to Cm34 does not significantly increase the stability of tRNA(Trp) association with its complementary anticodon in tRNA(Pro). The observations are discussed in the light of inter- and intra-strand stacking interactions among the anticodon triplets and with the purine base adjacent to them, and of possible biological implications.

Anticodon↗