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At least 973 records · Page 54Linked to original sources
PubMed1975

Structure-activity relationships in tryptophanyl transfer ribonucleic acid synthetase from beef pancreas. Influence of the alkylation of the sulfhydryl groups on the dimer-monomer equilibrium.

Upon reaction with N-ethylmaleimide, tryptophanyl-tRNA synthetase from beef pancreas dissociates into subunits. At pH7, the rate of the dissociation is close to both the reaction rate of the buried--SH groups and the rate of inactivation (Iborra, F., Mourgeon, G., Labouesse B., and Labouesse, J. (1973) Eur. J. Biochem. 39, 547-556). The pH and enzyme concnetration dependences of the reaction rate of the 16 cysteinyl residues of the enzyme as well as that of its inactivation support the idea that inactivation by alkylation of the--SH groups is due essentially to the dissociation of the protein into inactive subunits and not to the chemical blocking of a catalytic residue. This is confirmed by the independence on N-ethylmaleimide concentration of the reaction of the buried--SH groups and of the inactivation of the enzyme at high N-ethylmaleimide concentration. The dissociation becomes in this case the rate-limiting step of the chemical reaction. The monomeric structure is stabilized by the blocking of the--SH groups exposed during the dissociation. The dissociation constant of the dimeric enzyme is progressively increased during the alkylation. The tightness of the associated structure depends on the protonation of groups titrating between pH 7 and pH 9.

Amino Acyl-tRNA Synthetases↗
PubMed2000

NMR evidence for progressive stabilization of native-like structure upon aggregation of acid-denatured LysN.

The acid-denatured form of the protein LysN aggregates reversibly at pH 2.0. The strength of self-association increases with increasing Cl(-) anion concentration. At low concentrations of protein or Cl(-) anion, resonances of denatured LysN are in slow exchange with a minor form of the protein, which shows native-like NMR chemical shifts. The minor native-like resonances increase in intensity with increasing protein concentration, demonstrating that a native-like monomer fold is stabilized on aggregation of the acid-denatured protein. At high concentrations of protein or Cl(-) anion, interconversion between the major and minor resonances appears to shift from slow to intermediate exchange on the NMR timescale. NMR line-broadening is more pronounced for the major resonances of the denatured protein, which show sigmoidal decay curves with increasing Cl(-) concentration. The mid-points of the decay curves for residues in different parts of the molecule are non-coincident. We propose that differences in the NMR line-broadening transitions of individual residues reflect a stepwise stabilization of native-like structure on aggregation, starting with the segments of the protein that form the initial association interface. The resonances of the denatured protein with the greatest sensitivity to self-association correspond roughly to those that are most perturbed in the native protein on binding of the natural substrate tRNA(Lys). This suggests that the hydrophobic surfaces that promote intermolecular misfolding of acid-denatured LysN, may resemble those used for substrate binding by the native protein.

Acids↗
PubMed1993

A general two-metal-ion mechanism for catalytic RNA.

A mechanism is proposed for the RNA-catalyzed reactions involved in RNA splicing and RNase P hydrolysis of precursor tRNA. The mechanism postulates that chemical catalysis is facilitated by two divalent metal ions 3.9 A apart, as in phosphoryl transfer reactions catalyzed by protein enzymes, such as the 3',5'-exonuclease of Escherichia coli DNA polymerase I. One metal ion activates the attacking water or sugar hydroxyl, while the other coordinates and stabilizes the oxyanion leaving group. Both ions act as Lewis acids and stabilize the expected pentacovalent transition state. The symmetry of a two-metal-ion catalytic site fits well with the known reaction pathway of group I self-splicing introns and can also be reconciled with emerging data on group II self-splicing introns, the spliceosome, and RNase P. The role of the RNA is to position the two catalytic metal ions and properly orient the substrates via three specific binding sites.

Catalysis↗
PubMed2004

Three properties of the hepatitis C virus RNA genome related to antiviral strategies based on RNA-therapeutics: variability, structural conformation and tRNA mimicry.

The concept of using RNA molecules as therapeutic agents is receiving increasing attention by basic science and pharmaceutical research. Over the past five years, a number of clinical trials have been initiated to evaluate the efficacy and safety of several RNA agents for the treatment of a range of conditions from cancer to infectious disease. From a molecular biology perspective, two main factors are implicated in RNA therapeutics against pathogenic RNAs: i/ The activity, stability and delivery of the inactivating agent (ribozyme, RNase P, "decoy" RNA, aptamer, small interfering-RNA) and its co-localisation with the target; and ii/ The properties of the RNA substrate, which, in the case of an RNA virus, most likely limit the effectiveness of the inactivating agent. The main reasons are the limited size of the viral genome and the restrictions imposed by the RNA structure and variations at the target. In the first section of this article we review three properties of the HCV RNA genome, from primary sequence to tertiary structure, which imply restrictions and opportunities for RNA-based treatment. In the second section, we briefly describe several of the RNA-based therapeutic strategies against HCV now under development.

5' Untranslated Regions↗
PubMed2000

A homogeneous method to measure aminoacyl-tRNA synthetase aminoacylation activity using scintillation proximity assay technology.

A new method to measure the aminoacylation of tRNA based upon the use of the scintillation proximity assay (SPA) technology has been developed. The assay detects incorporation of radiolabeled amino acids into cognate tRNA, catalyzed by a specific aminoacyl-tRNA synthetase (aaRS). Under acidic conditions, uncoated yttrium silicate SPA beads were found to bind tRNA aggregates, while the radiolabeled amino acid substrate remains in solution, resulting in good signal discrimination of these two species in the absence of any separation steps. The usefulness of this approach was demonstrated by measurement of steady-state kinetic constants and inhibitor binding constants for a range of aaRS enzymes in comparison with data from standard, trichloroacetic acid-precipitation-based assays. In all cases, the data were quantitatively comparable. Although the radioisotopic counting efficiency of the SPA method was less than that of standard liquid scintillation counting, the statistical performance (i.e., signal to background, variability, stability) of the SPA assays was at least equivalent to the separation-based methods. The assay was also shown to work well in miniaturized 384-well microtiter plate formats, resulting in considerable reagent savings. In summary, a new method to characterize aaRS activity is described that is faster and more amenable to high-throughput screening than traditional methods.

Acylation↗
PubMed1997

Stability of HIV type 1 proviral genomes that contain two distinct primer-binding sites.

The initiation of human immunodeficiency virus type 1 (HIV-1) reverse transcription occurs by the extension of a tRNALys,3 positioned at an 18-nucleotide sequence in the RNA genome referred to as the primer-binding site (PBS). We have found that mutations within the PBS and a region upstream in U5, designated the A loop, influenced the selection of the tRNA primer used to initiate reverse transcription. Surprisingly, a proviral genome that contained a PBS and A loop complementary to tRNAPro resulted in the generation of viruses that contained two PBSs within the same genome: one of the PBSs in the virus was complementary to tRNALys,3 while the second PBS was complementary to tRNAIle, tRNAPro, or tRNALys,3. There were 14 nucleotides separating the two PBSs in the viral genome. In the current study, DNA encompassing U5 and the dual PBS complementary to the different tRNAs were amplified by PCR and exchanged for the corresponding region in an infectious HIV-1 clone, HXB2. Transfection of the different proviruses into cells resulted in the production of viruses that were infectious as determined by coculture with SupT1 cells. PCR was used to amplify the PBS regions from the different proviral DNAs followed by DNA sequencing of individual PCR clones. Proviruses containing the dual PBS complementary to tRNALys,3 and tRNAIle stably maintained the dual PBS complementary to both of these tRNAs following in vitro culture, although we noted consistent G-to-T and AA-to-GG substitutions in the 14-nucleotide region between the PBSs. The viruses derived from genomes that contained the dual PBS complementary to tRNALys,3 and tRNAPro also maintained both PBSs following in vitro culture; a single mutation was noted after in vitro culture in the 14-nucleotide region between the PBSs, which changed a consensus integration site (CA dinucleotide) prior to the PBS complementary to tRNAPro. In contrast, the proviral genomes containing the dual PBS complementary to tRNALys,3 were not stable and reverted back to a single PBS complementary to tRNALys,3. The results of our studies suggest that only the 5'-proximal PBS has been used to initiate reverse transcription. On the basis of our results, a mechanism is proposed for the generation of a dual PBS, which provides new insights into HIV-1 reverse transcription.

Animals↗
PubMed1980

Cultured animal cells exposed to amino acid analogues or puromycin rapidly synthesize several polypeptides.

Four major acidic polypeptides, with molecular weights of 88, 72, 71, and 23 thousand, and minor polypeptides with molecular weights of 110, 50, 38, and 30 thousand rapidly accumulated in cultured chick embryo (CE) cells which were exposed for three hours to the arginine analogue canavanine. P110, P88, P71,72, and P23 had unique peptide maps. Evidence of a 27,000 dalton precursor to P23 was obtained. The analogue-stimulated proteins were not related to another set of inducible avian polypeptides known as the glucose-regulated proteins. In mammalian cells, the rate of accumulation of several polypeptides, which were similar in size to the avian proteins, sharply increased after canavanine treatment. Proteins with the same electrophoretic mobilities, isoelectric points, and peptide maps as the analogue-stimulated proteins were expressed at low levels in untreated cultures. To determine the time courses of the canavanine-mediated increases in protein accumulation and the recovery of protein metabolism after analogue treatment, radioactively labeled proteins were extracted from CE cells and analyzed on SDS-polyacrylamide gels. In cultures exposed to canavanine, the rates of accumulation of P88 and P71,72 increased from basal to new plateau levels in about 1.5 hours, while P23 required about 2.5 hours. When added with the analogue, actinomycin D and cordycepin blocked the increases in protein accumulation. These inhibitors also blocked the rapid decline in the rates of accumulation of the enhanced proteins which occurred after removal of canavanine. Studies of the matabolic stability of the enhanced proteins indicated that the changes in their accumulation were caused by alterations in their rates of synthesis. Thus, the analogue-mediated response fulfilled several of the criteria for inducible eucaryotic gene expression. The amino acid analogue p-fluorophenylalanine and the chain-terminating analogue of amino acyl-tRNA puromycin stimulated the synthesis of the same set of proteins induced by canavanine. The enhanced synthesis of these proteins appeared to be a cellular response to either the presence or catabolism of abnormal proteins and puromycyl peptides.

Amino Acids↗
PubMed2004

Folding of small RNAs displaying the GNC base-pattern: implications for the self-organization of the genetic system.

Theoretical arguments and statistical analyses of present-day coding sequences have long suggested the existence of primitive patterns in RNA sequences, which were thought to have been predominant at the time of the origin of the genetic code. The main propositions were centered around the base-patterns GNC and RNY, where R = A or G , Y = C or U and N = A, G, C or U. A theoretical model of the primitive process of translation explaining the origin of this type of pattern was recently published in the Journal of Theoretical Biology. On the basis of this previous analysis, and on physico-chemical evidence supporting the idea of the GNC base-pattern as the most primitive one, the present paper shows the results of folding simulations of small RNA strands displaying this pattern, which enabled us to specify the characteristics of the suggested primitive form of tRNA. This analysis is notably based on a conjecture of Eigen and Schuster of an early structural (or pattern) similarity between mRNA and tRNA, and, more specifically, of a "joint function of messenger and adaptor". Working with this conjecture, we show that the convergence of the primitive pool of RNAs toward a system containing a high proportion of sequences displaying the GNC base-pattern (according to the evolutionary model) is accompanied by a significant gain in stability of the translation process. In particular, it is demonstrated how the reading frame would be automatically discriminated without the presence of a start codon.

Animals↗
PubMed2000

Synthesis and properties of uniquely modified oligoribonucleotides: yeast tRNA(Phe) fragments with 6-methyluridine and 5,6-dimethyluridine at site-specific positions.

The phosphoramidites of 6-methyluridine and 5,6-dimethyluridine were synthesized and the modified uridines site-selectively incorporated into heptadecamers corresponding in sequence to the yeast tRNA(Phe) anticodon and TpsiC domains. The oligoribonucleotides were characterized by NMR, MALDI-TOF MS and UV-monitored thermal denaturations. The 6-methylated uridines retained the syn conformation at the polymer level and in each sequence location destabilized the RNAs compared to that of the unmodified RNA. The decrease in RNA duplex stability is predictable. However, loss of stability when the modified uridine is in a loop is sequence context dependent, and can not, at this time, be predicted from the location in the loop.

Anticodon↗
PubMed2005

Evaluation of the conformational switch model for alfalfa mosaic virus RNA replication.

Key elements of the conformational switch model describing regulation of alfalfa mosaic virus (AMV) replication (R. C. Olsthoorn, S. Mertens, F. T. Brederode, and J. F. Bol, EMBO J. 18:4856-4864, 1999) have been tested using biochemical assays and functional studies in nontransgenic protoplasts. Although comparative sequence analysis suggests that the 3' untranslated regions of AMV and ilarvirus RNAs have the potential to fold into pseudoknots, we were unable to confirm that a proposed pseudoknot forms or has a functional role in regulating coat protein-RNA binding or viral RNA replication. Published work has suggested that the pseudoknot is part of a tRNA-like structure (TLS); however, we argue that the canonical sequence and functional features that define the TLS are absent. We suggest here that the absence of the TLS correlates directly with the distinctive requirement for coat protein to activate replication in these viruses. Experimental data are evidence that elevated magnesium concentrations proposed to stabilize the pseudoknot structure do not block coat protein binding. Additionally, covarying nucleotide changes proposed to reestablish pseudoknot pairings do not rescue replication. Furthermore, as described in the accompanying paper (L. M. Guogas, S. M. Laforest, and L. Gehrke, J. Virol. 79:5752-5761, 2005), coat protein is not, by definition, inhibitory to minus-strand RNA synthesis. Rather, the activation of viral RNA replication by coat protein is shown to be concentration dependent. We describe the 3' organization model as an alternate model of AMV replication that offers an improved fit to the available data.

Alfalfa mosaic virus↗
PubMed1996

Processing of the leader mRNA plays a major role in the induction of thrS expression following threonine starvation in Bacillus subtilis.

The threonyl-tRNA synthetase gene, thrS, is a member of a family of Gram-positive genes that are induced following starvation for the corresponding amino acid by a transcriptional antitermination mechanism involving the cognate uncharged tRNA. Here we show that an additional level of complexity exists in the control of the thrS gene with the mapping of an mRNA processing site just upstream of the transcription terminator in the thrS leader region. The processed RNA is significantly more stable than the full-length transcript. Under nonstarvation conditions, or following starvation for an amino acid other than threonine, the full-length thrS mRNA is more abundant than the processed transcript. However, following starvation for threonine, the thrS mRNA exists primarily in its cleaved form. This can partly be attributed to an increased processing efficiency following threonine starvation, and partly to a further, nonspecific increase in the stability of the processed transcript under starvation conditions. The increased stability of the processed RNA contributes significantly to the levels of functional RNA observed under threonine starvation conditions, previously attributed solely to antitermination. Finally, we show that processing is likely to occur upstream of the terminator in the leader regions of at least four other genes of this family, suggesting a widespread conservation of this phenomenon in their control.

Amino Acyl-tRNA Synthetases↗
PubMed1996

Structure of a hexanucleotide RNA hairpin loop conserved in ribosomal RNAs.

The structure of 5'-pppGGAC(GUAAUA)GUCC has been deduced from NMR data. The six-nucleotide hairpin loop is highly conserved in large subunit ribosomal RNA and confers unusual stability on RNA hairpins. Standard assignment methods, including through-bond strategies for backbone protons, were used to assign all 31P resonances and all but two of the non-exchangeable protons. The model calculated by restrained molecular dynamics shows that the loop adopts a specific structure stabilized by five non-canonical hydrogen bonds. An edge-to-edge G-A pair (hydrogen bonds G5 N3-A10 NH6, G5 H2-A1O N7 and A10 NH6-G5 O2') closes the hairpin loop. A trans 5' P-O bond at A7 reverses the direction of the backbone and is stabilized by two base-backbone hydrogen bonds (U6 NH3-U9 OP and U6 O2'-A8 N7). The only unstructured part of the loop is U9, which is completely unstacked and also the only phylogenetically variable position. The U6-A7-A8 "U-turn" reproduces hydrogen bonds and backbone torsion seen in the tRNA anticodon loop and TTpsiC loop, and in an internal loop of the hammerhead ribozyme. G-A is a common closing mismatch in ribosomal RNA hairpin loops. The U-turn and G-A pair found in this hexaloop structure may therefore be common structural units of RNA hairpin loops.

Base Sequence↗
PubMed1980

Insertions of Tn 10 into an E. coli ribosomal RNA operon are incompletely polar.

Insertions of the transposon Tn 10 have been obtained in an E. coli ribosomal RNA operon (rrnX). These insertions were used to determine whether present models of polarity are sufficient to explain transcriptional properties of operon which are transcribed but not translated. Most models of polarity suggest that transcription of mRNA requires simultaneous translation of nascent mRNA, or else premature termination of transcription will result. This model as stated does not encompass rrn operons. In this study three Tn 10 insertions into rrnX were characterized. All three Tn 10 insertions do not completely eliminate in vivo transcription of the most distal tRNA gene of rrnX. In ultraviolet-irradiated cells one Tn 10 insertion reduces synthesis of downstream portions of rrnX by at most 2--3 fold. Two other insertions may have more extensive polar effects, although the exact level of polarity is difficult to evaluate because these Tn 10 insertions may affect RNA maturation and stability.

DNA Transposable Elements↗
PubMed2006

Interactions of designer antibiotics and the bacterial ribosomal aminoacyl-tRNA site.

The X-ray crystal structures for the complexes of three designer antibiotics, compounds 1, 2, and 3, bound to two models for the ribosomal aminoacyl-tRNA site (A site) at 2.5-3.0 Angstroms resolution and that of neamine at 2.8 Angstroms resolution are described. Furthermore, the complex of antibiotic 1 bound to the A site in the entire 30S ribosomal subunit of Thermus thermophilus is reported at 3.8 Angstroms resolution. Molecular dynamics simulations revealed that the designer compounds provide additional stability to bases A1492 and A1493 in their extrahelical forms. Snapshots from the simulations were used for free energy calculations, which revealed that van der Waals and hydrophobic effects were the driving forces behind the binding of designer antibiotic 3 when compared to the parental neamine.

Anti-Bacterial Agents↗
PubMed2006

Structure and function of eukaryotic Ribonuclease P RNA.

Ribonuclease P (RNase P) is the ribonucleoprotein endonuclease that processes the 5' ends of precursor tRNAs. Bacterial and eukaryal RNase P RNAs had the same primordial ancestor; however, they were molded differently by evolution. RNase P RNAs of eukaryotes, in contrast to bacterial RNAs, are not catalytically active in vitro without proteins. By comparing the bacterial and eukaryal RNAs, we can begin to understand the transitions made between the RNA and protein-dominated worlds. We report, based on crosslinking studies, that eukaryal RNAs, although catalytically inactive alone, fold into functional forms and specifically bind tRNA even in the absence of proteins. Based on the crosslinking results and crystal structures of bacterial RNAs, we develop a tertiary structure model of the eukaryal RNase P RNA. The eukaryal RNA contains a core structure similar to the bacterial RNA but lacks specific features that in bacterial RNAs contribute to catalysis and global stability of tertiary structure.

Azides↗
PubMed1979

Proton nuclear magnetic resonance of minor nucleosides in yeast phenylalanine transfer ribonucleic acid. Conformational changes as a consequence of aminoacylation, removal of the Y base, and codon--anticodon interaction.

The assignments of the resonances of the methyl and methylene groups belonging to the residues dihydro-uridine-16 and -17 (C5 and C6), dimethylguanosine-26, N-2-methylguanosine-10, and 7-methylguanosine-46 of yeast tRNAPhe at low temperature are reported. Observing the high-field proton NMR spectral region at different temperatures, the effects of aminoacylation, removal of the Y base, and codon-anticodon interaction on the tertiary structure of yeast tRNAPhe were investigated. The following are the results of this study. (1) The two dihydrouridine residues of tRNAPhe have different environments in aqueous solution: dihydro-uridine-16 is more shielded than dihydrouridine-17. (2) The ribothymidine residue from the fragment (47--76) of yeast tRNAPhe and from a tRNA with a partially disrupted structure exhibits multiple conformations arising from different stacking modes between the ribothymidine-54 and the guanosine-53 residue. (3) Upon aminoacylation the type of guanosine-53 interaction with ribothymidine-54 in the tRNAPhe changes. (4) Removal of the Y base from the anticodon loop of yeast tRNAPhe weakens the thermal stability of the tertiary interactions. (5) The interaction of two complementary anticodons in the absence of proteins and of ribosomes results in stabilization of the tertiary structure. Codon-anticodon interaction dependent rearrangement of the tertiary structure of yeast tRNAPhe was not observed. The spin-lattice relaxation times of the methyl and methylene groups of the minor nucleosides in yeast tRNAPhe demonstrate that the minor nucleosides undergo rotational reorientation (tau c) in the nano-second range. The observed differences in these tau c values indicate a similarity of structure of tRNAPhe in solution and in crystalline form.

Anticodon↗
PubMed1994

The effect of elevated plasma phenylalanine levels on protein synthesis rates in adult rat brain.

Increasing the plasma phenylalanine concentration to levels as high as 0.560-0.870 mM (over ten times normal levels) had no detectable effect on the rate of brain protein synthesis in adult rats. The average rates for 7-week-old rats were: valine, 0.58 +/- 0.05%/h, phenylalanine, 0.59 +/- 0.06%/h, and tyrosine, 0.60 +/- 0.09%/h, or 0.59 +/- 0.06%/h overall. Synthesis rates calculated on the basis of the specific activity of the tRNA-bound amino acid were slightly lower (4% lower for phenylalanine) than those based on the brain free amino acid pool. Similarly, the specific activities of valine and phenylalanine in microdialysis fluid from striatum were practically the same as those in the brain free amino acid pool. Thus the specific activities of the valine and phenylalanine brain free pools are good measures of the precursor specific activity for protein synthesis. In any event, synthesis rates, whether based on the specific activities of the amino acids in the brain free pool or those bound to tRNA, were unaffected by elevated levels of plasma phenylalanine. Brain protein synthesis rates measured after the administration of quite large doses of phenylalanine (> 1.5 mumol/g) or valine (15 mumol/g) were in agreement (0.62 +/- 0.01 and 0.65 +/- 0.01%/h respectively) with the rates determined with infusions of trace amounts of amino acids. Thus the technique of stabilizing precursor-specific activity, and pushing values in the brain close to those of the plasma, by the administration of large quantities of precursor, appears to be valid.

Animals↗
PubMed1999

Major rearrangements in the 70S ribosomal 3D structure caused by a conformational switch in 16S ribosomal RNA.

Dynamic changes in secondary structure of the 16S rRNA during the decoding of mRNA are visualized by three-dimensional cryo-electron microscopy of the 70S ribosome. Thermodynamically unstable base pairing of the 912-910 (CUC) nucleotides of the 16S RNA with two adjacent complementary regions at nucleotides 885-887 (GGG) and 888-890 (GAG) was stabilized in either of the two states by point mutations at positions 912 (C912G) and 885 (G885U). A wave of rearrangements can be traced arising from the switch in the three base pairs and involving functionally important regions in both subunits of the ribosome. This significantly affects the topography of the A-site tRNA-binding region on the 30S subunit and thereby explains changes in tRNA affinity for the ribosome and fidelity of decoding mRNA.

Base Sequence↗