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PubMed1991

Relation between functions and conformational characteristics of modified nucleosides found in tRNAs.

Conformational characteristics of N4-acetyl-2'-O-methylcytidine (ac4Cm), 5-methyl-2'-O-methylcytidine (m5Cm) and N2-dimethyl-2'-O-methylguanosine (m2(2)Gm) found in tRNAs from extremely thermophilic archaebacteria were analyzed by proton NMR spectroscopy. The 2'-O-methylation of ac4C, m5C and m2(2)G was found to stabilize the C3'-endo form and therefore cause "conformational rigidity". In particular, the ac4Cm was found to be extremely rigid due to additive effects of the N4-acetylation and 2'-O-methylation. Therefore, tRNAs from the extremely thermophilic archaebacteria use the base modifications in combination with the 2'-O-methylation, resulting in stabilization of the A-type conformation at specific positions in the tRNAs even at very high temperatures. In contrast, mesophile tRNAs use for a given site only one of these ribose and base modifications each of which is effective enough by itself at ordinary temperatures. These findings are consistent with our previous findings that roles of a variety of post-transcriptional modifications are to regulate the conformational rigidity/flexibility which is essential for the tRNA functions.

Archaea↗

PubMed1998

Complete kinetic mechanism of elongation factor Tu-dependent binding of aminoacyl-tRNA to the A site of the E. coli ribosome.

The kinetic mechanism of elongation factor Tu (EF-Tu)-dependent binding of Phe-tRNAPhe to the A site of poly(U)-programmed Escherichia coli ribosomes has been established by pre-steady-state kinetic experiments. Six steps were distinguished kinetically, and their elemental rate constants were determined either by global fitting, or directly by dissociation experiments. Initial binding to the ribosome of the ternary complex EF-Tu.GTP.Phe-tRNAPhe is rapid (k1 = 110 and 60/micromM/s at 10 and 5 mM Mg2+, 20 degreesC) and readily reversible (k-1 = 25 and 30/s). Subsequent codon recognition (k2 = 100 and 80/s) stabilizes the complex in an Mg2+-dependent manner (k-2 = 0.2 and 2/s). It induces the GTPase conformation of EF-Tu (k3 = 500 and 55/s), instantaneously followed by GTP hydrolysis. Subsequent steps are independent of Mg2+. The EF-Tu conformation switches from the GTP- to the GDP-bound form (k4 = 60/s), and Phe-tRNAPhe is released from EF-Tu.GDP. The accommodation of Phe-tRNAPhe in the A site (k5 = 8/s) takes place independently of EF-Tu and is followed instantaneously by peptide bond formation. The slowest step is dissociation of EF-Tu.GDP from the ribosome (k6 = 4/s). A characteristic feature of the mechanism is the existence of two conformational rearrangements which limit the rates of the subsequent chemical steps of A-site binding.

Codon↗

PubMed1999

Crystal structure of acceptor stem of tRNA(Ala) from Escherichia coli shows unique G.U wobble base pair at 1.16 A resolution.

The acceptor stem of Escherichia coli tRNA(Ala), rGGGGCUA.rUAGCUCC (ALAwt), contains the main identity element for the correct aminoacylation by the alanyl tRNA synthetase. The presence of a G3.U70 wobble base pair is essential for the specificity of this reaction, but there is a debate whether direct minor-groove contact with the 2-amino group of G3 or a distortion of the acceptor stem induced by the wobble pair is the critical feature recognized by the synthetase. We here report the structure analysis of ALAwt at near-atomic resolution using twinned crystals. The crystal lattice is stabilized by a novel strontium binding motif between two cis-diolic O3'-terminal riboses. The two independent molecules in the asymmetric unit of the crystal show overall A-RNA geometry. A comparison with the crystal structure of the G3-C70 mutant of the acceptor stem (ALA(C70)) determined at 1.4 A exhibits a modulation in ALAwt of helical twist and slide due to the wobble base pair, but no recognizable distortion of the helix fragment distant from the wobble base pair. We suggest that a highly conserved hydration pattern in both grooves around the G3.U70 wobble base pair may be functionally significant.

Base Pairing↗

PubMed1976

Multiple forms of histone acetyltransferases in the cytosol of calf endometrium.

The histone acetyltransferase (EC 2.3.1.-) activity of calf endometrium cytosol has been separated into three separate activities by stepwise chromatography on DEAE-cellulose. In addition to differential elution from the DEAE-cellulose, the three activities are differentiated by their pH optima, preferences for histone subfractions as substrates, and stability to heat denaturation. Peak I has an optimum of pH 8.7 and preferentially acetylates histones F2b and F3; Peak II has an optimum of pH 8.5, and preferentially acetylates histone F2al followed by histone F2b; Peak III has an optimum of pH 9.5, and had similar specificity to Peak II. Peak III is appreciably more stable at 60 degrees C than is Peak II. None of the peaks transferred acetate to other proteins tested or to tRNA. These studies suggest the presence of multiple histone acetyltransferases in tissue cytosols.

Acetates↗

PubMed1985

Functional selection and analysis of yeast centromeric DNA.

A direct selection procedure has been used to isolate 11 distinct yeast genomic DNA fragments that eliminate the extreme segregation bias characteristic of autonomously replicating yeast plasmids. The selection scheme takes advantage of the fact that the cloned ochre suppressing tRNA gene, SUP11, is lethal at high copy number and therefore causes cell death when present on an ARS plasmid that lacks a cis-acting partition function. Each of the cloned DNA sequences was mapped to specific yeast chromosomes by hybridization to chromosome-sized DNA molecules separated by alternating field electrophoresis. Ten of the cloned fragments correspond to chromosomal centromeres; one fragment corresponds to the cis-acting locus required for endogenous 2 mu plasmid stability. Nucleotide sequence comparison of the ten centromere DNAs gives a new picture of conserved centromere DNA elements.

Base Sequence↗

PubMed1994

Construction and characterization of lck- and fyn-specific tRNA: ribozyme chimeras.

Two src-family protein tyrosine kinases (PTKs), p56lck, and p59fyn, are thought to play an important role in the antigen-specific T cell receptor (TCR)/CD3-initiated signaling pathway, but their relative contribution to these events is not clearly defined. Here, we have explored the potential of catalytic RNA molecules, or ribozymes, as tools for selectively inhibiting expression of the corresponding target genes in T cells. Several lck- or fyn-specific hammerhead ribozymes were synthesized, cloned into a bacterial transcription vector, and found to display specific catalytic activity in vitro. In order to achieve stable high-level ribozyme expression in intact cells, selected ribozymes were subsequently cloned into a retroviral vector (DC-T5T) immediately downstream of a tRNA(met) transcription unit. Upon retroviral transduction of a human leukemic T cell line (Jurkat), two out of four chimeric tRNA:ribozymes, fyn-1 and lck-1, were stably expressed at levels of approximately 10,000 or approximately 25,000 copies/cell, respectively. Ribozyme expression was associated with a reduction of up to 80% (lck) or 61% (fyn) in endogenous target mRNA by comparison to the corresponding transcript levels in control clones transfected with vector alone. By contrast, expression of the corresponding target proteins was not reduced, suggesting a post-transcriptional compensatory mechanism that increases translation or stability of the p56lck and/or p59fyn proteins.

Base Sequence↗

PubMed1979

The binding of non-cognate Tyr-tRNATyr to poly(uridylic acid)-programmed Escherichia coli ribosomes.

The poly(U)-dependent binding of Tyr-tRNATyr to Escherichia coli ribosomes has been studied using a highly purified system. Binding is maximal at 10 mM magnesium acetate (up to 0.7 molecule Tyr-tRNATyr/ribosome), and requires the presence of elongation factor (EF) T (a mixture of EF-Ts and EF-Tu), GTP, NH4+ ions and an aminoglycoside antibiotic (streptomycin, neomycin B, kanamycin B or gentamicin C1a). Under limiting and up to saturating concentrations of EF-T, one molecule of GTP is hydrolyzed per molecule of Tyr-tRNATyr bound, suggesting that 'proof-reading' mechanisms involving the hydrolysis of GTP are inoperative in the presence of the antibiotics. Binding of Tyr-tRNATyr apparently takes place at the ribosomal acceptor site, since peptide bonds are readily formed with N-acetyl-Phe-tRNA prebound to the ribosomal donor site. In contrast to Phe-tRNAPhe binding, Tyr-tRNATyr binding is impaired by the omission of the 50-S subunit, the replacement of GTP by its non-hydrolyzable analogs guanyl-5'-yl methylene diphosphonate and guanyl-5'-yl iminodiphosphonate, and also by the presence of the antibiotic streptogramin A. This suggests that the correct interaction of Tyr-tRNATyr with the peptidyl transferase centre is essential for the stability of this ligand on the ribosome. Moreover, the aminoglycoside antibiotics are also necessary, even after the binding reaction is complete, to maintain Tyr-tRNATyr on the ribosome.

Aminoglycosides↗

PubMed1992

Tagetitoxin inhibition of RNA polymerase III transcription results from enhanced pausing at discrete sites and is template-dependent.

In yeast nuclear extracts, tagetitoxin inhibition of RNA polymerase III promoter-directed single- and multiple-round transcription is characterized by pausing or stalling of the elongation complex at several discrete points on the template. Paused ternary complexes isolated from tagetitoxin-inhibited reactions can be elongated to produce full-length RNA. The distribution of "tagetitoxin-enhanced" pause sites is distinct for each of the class III genes we have examined. These tagetitoxin-enhanced pause sites may also be intrinsic pause sites for the elongation complex. Tagetitoxin inhibition of in vitro transcription of the yeast SUP4 and SUP6 tRNA(Tyr) genes demonstrates template dependence and indicates that inhibition may occur after UMP incorporation. Factor-independent transcription by purified yeast RNA polymerase III can also be inhibited by tagetitoxin, and the degree of inhibition is template-dependent. Tagetitoxin may be most effective as an inhibitor under conditions where polymerase III tends to pause on the template. We propose that differences in tagetitoxin inhibition among class III genes may reflect differences in the number of stability of these pause sites.

Animals↗

PubMed1995

Properties of isolated domains of the elongation factor Tu from Thermus thermophilus HB8.

The relative contributions of the three domains of elongation factor Tu (EF-Tu) to the factor's function and thermal stability were established by dissecting the domains apart with recombination techniques. Domain I (EF-TuI), domains I/II (EF-TuI/II) and domain III (EF-TuIII) of the EF-Tu from Thermus thermophilus HB8 comprising the amino acids 1-211, 1-312 and 317-405, respectively, were overproduced in Escherichia coli and purified. A polypeptide consisting of domain II and III (EF-TuII/III) was prepared by limited proteolysis of native EF-Tu with V8 protease from Staphylococcus aureus [Peter, M. E., Reiser, C. O. A., Schirmer, N. K., Kiefhaber, T., Ott, G., Grillenbeck, N. W. & Sprinzl, M. (1990) Nucleic Acids Res. 18, 6889-6893]. As determined by circular dichroism spectrometry, the isolated domains have the secondary structure elements found in the native EF-Tu. GTP and GDP binding as well as GTPase activity are maintained by the EF-TuI and EF-TuI/II; however, the rate of GDP dissociation from EF-TuI . GDP and EF-TuI/II . GDP complex is increased as compared to native EF-Tu . GDP, reflecting a constraint imposed by domain III on the ability to release the nucleotide from its binding pocket located in domain I. A weak interaction of Tyr-tRNATyr with the EF-TuI . GTP suggests that domain I provides a part of the structure interacting with aminoacyl-tRNA. The domain III is capable of regulating the rate of GTPase in EF-Tu, since the polypeptide consisting only of domains I/II has a 39-fold higher intrinsic GTPase compared to the native EF-Tu. No in vitro poly(U)-dependent poly(Phe) synthesis was detectable with a mixture of equimolar amounts of domains I/II and domain III, demonstrating the necessity of covalent linkage between the domains of EF-Tu for polypeptide synthesis. In contrast to native EF-Tu and EF-TuII/III, EF-TuI and, to a lesser extent the polypeptide consisting of domains I/II, are unstable at elevated temperatures. This indicates that domains II/III strongly contribute to the thermal stability of this T. thermophilus EF-Tu. Deletion of amino acid residues 181-190 from domain I of T. thermophilus EF-Tu decreases the thermostability to that of EF-Tu from E. coli, which does not have these residues. Interdomain interactions must be important for the stabilisation of the structure of domain I, since isolated T. thermophilus EF-TuI is thermolabile despite the presence of the 181-190 loop.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1997

Mutation accumulation in nuclear, organelle, and prokaryotic transfer RNA genes.

A comparative analysis of the transfer RNA genes in the genomes of the major kingdoms of eukaryotes and prokaryotes leads to the general conclusion that the rate of evolution of organelle tRNA genes is typically equal to of greater than that of their nuclear counterparts. Situations where this is not the case, most notably in vascular plants, are attributable to an elevated mutation rate in the nuclear genome. Through a comparison of rates of mutation with rates of nucleotide substitution, it is shown that there is a reduction in the efficiency of selection on new mutations in organelle genes. Numerous lines of evidence, including observed reductions in stem duplex stability and changes in loop sizes, suggest that the excess changes observed in the organelle genes are mildly deleterious. Uniparental inheritance of organelles causes a reduction in the efficiency of selection through the joint effects of an increase in linkage disequilibrium and a decrease in effective population size. These results provide molecular support for the idea that asexually propagating genomes are subject to long-term, gradual fitness loss and raise questions about the role of organelle mutations in the long-term survival of major phylogenetic lineages.

Animal Population Groups↗

PubMed2004

Crystal structure of leucyl-tRNA synthetase from the archaeon Pyrococcus horikoshii reveals a novel editing domain orientation.

The editing domains of the closely homologous leucyl, isoleucyl, and valyl-tRNA synthetases (LeuRS, IleRS, and ValRS, respectively) contribute to accurate aminoacylation, by hydrolyzing misformed non-cognate aminoacyl-tRNAs. The editing domain is inserted at the same point of the sequence in IleRS, ValRS, and the archaeal/eukaryal LeuRS, but at a distinct point in the bacterial LeuRS. Here, we showed that LeuRS from the archaeon Pyrococcus horikoshii has editing activity against the nearly cognate isoleucine. The conserved Asp332 in the editing domain is crucial for this activity. A deletion mutant lacking the C-terminal region has only negligible aminoacylation activity, but retains the full activity of adenylate synthesis and editing. We determined the crystal structure of this editing-active, truncated form of P.horikoshii LeuRS at 2.1 A resolution. The structure revealed that it has a novel editing domain orientation. The editing domain of P.horikoshii LeuRS is rotated by approximately 180 degrees (rotational state II), with the two-beta-stranded linker untwisted by a half-turn, as compared to those in IleRS and ValRS (rotational state I). This editing domain rotational state in the archaeal LeuRS is similar to that in the bacterial LeuRS. However, because of the insertion point difference, the orientation of the editing domain relative to the enzyme core in the archaeal LeuRS differs completely from that in the bacterial LeuRS. An insertion region specific to the archaeal/eukaryal LeuRS editing domains interacts with the enzyme core and stabilizes the unique orientation. Thus, we established that there are three types of editing domain orientations relative to the enzyme core, depending on the combination of the editing domain insertion point (i or ii) and the rotational state (I or II): [i, I] for IleRS and ValRS, [ii, II] for the bacterial LeuRS, and now [i, II] for the archaeal/eukaryal LeuRS.

Amination↗

PubMed2006

RNase Z in Escherichia coli plays a significant role in mRNA decay.

Genetic and biochemical analysis of RNase Z in eukaryotes, such as Arabadopsis thaliana, and prokaryotes like Bacillus subtilis have demonstrated that this endoribonuclease is essential for the maturation of tRNA precursors that do not contain a chromosomally encoded CCA determinant. As all Escherichia coli tRNA transcripts have chromosomally encoded CCA determinants, the function of its putative RNase Z homologue, the product of the elaC gene, is not clear. Here we demonstrate that the E. coli ElaC protein (RNase Z) endonucleolytically processes B. subtilis tRNA precursors lacking a CCA determinant both in vivo and in vitro. More importantly, E. coli RNase Z plays a significant role in mRNA decay, a previously unidentified activity for the enzyme. The purified RNase Z protein cleaves the rpsT mRNA at locations distinct from those obtained with RNase E. As expected, under physiological conditions E. coli and B. subtilis tRNA precursors containing a CCA determinant are not substrates. These results suggest a potentially important new role for the RNase Z family of proteins in RNA metabolism, particularly in organisms lacking RNase E.

Bacillus subtilis↗

PubMed1997

Effects of Mg2+, K+, and H+ on an equilibrium between alternative conformations of an RNA pseudoknot.

A complex pseudoknot structure surrounds the first ribosome initiation site in the Escherichia coli alpha mRNA and mediates its regulation by ribosomal protein S4. A 112 nt RNA fragment containing this pseudoknot exists in two conformations that are resolvable by gel electrophoresis below room temperature. Between 30 degrees C and 45 degrees C the conformers reach thermodynamic equilibrium on a time scale ranging from one hour to one minute, and the interconversion between conformers is linked to H+, K+ and Mg2+ concentrations. Mg2+ favors formation of the "fast" electrophoretic form: a single Mg2+ is bound in the rate-limiting step, followed by cooperative binding of approximately 1.7 additional ions. Binding of the latter ions provides most of the favorable free energy for the reaction. However, the "slow" form binds about the same number of Mg ions, albeit more weakly, so that saturating Mg2+ concentrations drive the equilibrium to only approximatley 70% fast form. A single H+ is taken up in the switch to the "slow" conformer, which has apparent pK approximately 5.9; low pH also stabilizes part of the pseudoknot structure melting at approximately 62 degrees C. Mg2+ and H+ appear to direct alpha mRNA folding by relatively small (10 to 100-fold) differences in their affinities for alternative conformers. K+ has very little effect on the conformational equilibrium, but at high concentrations accelerates interconversion between the conformers. The alpha mRNA conformational switch is similar in its slow kinetics, large activation energy, and Mg2+ dependence of the equilibrium constant to slow steps in the folding of tRNA, group I introns, and RNase P RNA tertiary structures, though it differs from these in the association of a single Mg2+ with the rate-limiting step.

Base Sequence↗

PubMed1998

Presence and location of modified nucleotides in Escherichia coli tmRNA: structural mimicry with tRNA acceptor branches.

Escherichia coli tmRNA functions uniquely as both tRNA and mRNA and possesses structural elements similar to canonical tRNAs. To test whether this mimicry extends to post-transcriptional modification, the technique of combined liquid chromatography/ electrospray ionization mass spectrometry (LC/ESIMS) and sequence data were used to determine the molecular masses of all oligonucleotides produced by RNase T1 hydrolysis with a mean error of 0.1 Da. Thus, this allowed for the detection, chemical characterization and sequence placement of modified nucleotides which produced a change in mass. Also, chemical modifications were used to locate mass-silent modifications. The native E.coli tmRNA contains two modified nucleosides, 5-methyluridine and pseudouridine. Both modifications are located within the proposed tRNA-like domain, in a seven-nucleotide loop mimicking the conserved sequence of T loops in canonical tRNAs. Although tmRNA acceptor branches (acceptor stem and T stem-loop) utilize different architectural rules than those of canonical tRNAs, their conformations in solution may be very similar. A comparative structural and functional analysis of unmodified tmRNA made by in vitro transcription and native E.coli tmRNA suggests that one or both of these post-transcriptional modifications may be required for optimal stability of the acceptor branch which is needed for efficient aminoacylation.

Base Composition↗

PubMed1997

Overexpression of human release factor 1 alone has an antisuppressor effect in human cells.

Two eukaryotic proteins involved in translation termination have recently been characterized in in vitro experiments. Eukaryotic release factor 1 (eRF1) catalyzes the release of the polypeptide chain without any stop codon specificity. The GTP-binding protein eRF3 confers GTP dependence to the termination process and stimulates eRF1 activity. We used tRNA-mediated nonsense suppression at different stop codons in a cat reporter gene to analyze the polypeptide chain release factor activities of the human eRF1 and eRF3 proteins overexpressed in human cells. In a chloramphenicol acetyltransferase assay, we measured the competition between the suppressor tRNA and the human release factors when a stop codon was present in the ribosomal A site. Whatever the stop codon (UAA, UAG, or UGA) present in the cat open reading frame, the overexpression of human eRF1 alone markedly decreased translational readthrough by suppressor tRNA. Thus, like the procaryotic release factors RF1 and RF2 in Escherichia coli, eRF1 seems to have an intrinsic antisuppressor activity in human cells. Levels of antisuppression of overexpression of both eRF3 and eRF1 were almost the same as those of overexpression of eRF1 alone, suggesting that eRF1-eRF3 complex-mediated termination may be controlled by the expression level of eRF1. Surprisingly, when overexpressed alone, eRF3 had an inhibitory effect on cat gene expression. The results of cat mRNA stability studies suggest that eRF3 inhibits gene expression at the transcriptional level. This indicates that in vivo, eRF3 may perform other functions, including the stimulation of eRF1 activity.

Blotting, Northern↗

PubMed2000

Synthesis and properties of 2'-O-methyl-2-thiouridine and oligoribonucleotides containing 2'-O-methyl-2-thiouridine.

A new method for the synthesis of 2'-O-methyl-2-thiouridine (s2Um) found in thermophilic bacterial tRNA was developed. Structural properties of s2Um and s2Um(p)U were studied by using 1H NMR spectroscopy. A modified nonaribonucleotide (RNA*: 5'-CGUUs2UmUUGC-3') was synthesized to study the base-recognition ability of s2Um in formation of RNA-RNA and RNA DNA duplexes. The UV melting experiments revealed that RNA*-RNA and RNA*-DNA duplexes having an s2U-A base pair are more stable than those having a U-A base pair. On the contrary, the thermal stability of RNA*-RNA and RNA*-DNA duplexes having an s2U-G wobble base pair was much lower than that of the unmodified duplexes having a natural U-G base pair. It is concluded that s2Um has higher selectivity toward A over G than unmodified U.

Archaea↗

PubMed1995

Site-directed mutagenesis with an expanded genetic code.

A biosynthetic method has been developed that makes possible the site-specific incorporation of a large number of amino acids and analogues within proteins. In this approach, an amber suppressor tRNA chemically aminoacylated with the desired amino acid incorporates this amino acid site specifically into a protein in response to an amber codon introduced at the corresponding position in the protein's DNA sequence. Using this method, precise changes within a protein can be made to address detailed structure-function questions. A series of fluorinated tyrosine analogues and linear, branched, and cyclic hydrophobic amino acids have been used to determine the impact of hydrogen bonding and hydrophobic packing, respectively, on protein stability. Glutamate analogues and conformationally restricted amino acids have been used to probe the mechanisms of staphylococcal nuclease and ras. In addition, this technique has been used to construct photocaged proteins and proteins containing photoaffinity labels, spin labels, and isotopic labels at specific positions in the protein sequence suitable for biophysical studies.

Amino Acids↗

PubMedDate not supplied

On the reactivity of pyridoxal-5'-phosphate with yeast tRNAPhe and tRNATyr.

Yeast tRNAPhe and tRNATyr were reacted with the fluorescent reagent pyridoxal-5'-phosphate and the modified tRNAs were analysed with respect to the number and position of modified nucleosides and with respect to aminoacylation. a) Following the intrinsic fluorescence of pyridoxal-5'-phosphate, the treatment of tRNATyr with increasing amounts of pyridoxal-5'-phosphate revealed about 50 mol or reagent or a even higher number bound per one mol of tRNATyr. After borohydride reduction (in order to stabilize the linkage) of this modified tRNATyr and purification with reverse phase chromatography a modified tRNATyr was obtained carrying about 2 mol of the reagent. b) Both tRNATyr and tRNAPhe treated with pyridoxal-5'-phosphate and reduced exhibited almost unchanged aminoacylation as compared to the unmodified tRNAs. c) Pyridoxal-5'-phosphate treated and reduced tRNAPhe and tRNATyr were digested with ribonuclease T1 and the resulting oligonucleotides were separated. However, no fluorescent oligonucleotide and no difference to an oligonucleotide pattern obtained from unmodified tRNA were observed. Thus, pyridoxal-5'-phosphate might have been bound to the highly purified yeast tRNAPhe and tRNATyr samples either via an unstable linkage or not covalently. This result is controversial with respect to the specific reaction of pyridoxal-5'-phosphate with unfractionated tRNAs from colon carcinoma and tRNAs from E. coli as reported in the literature.

Chemistry, Organic↗