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At least 883 records · Page 49Linked to original sources
PubMed1998

Modulation of cellular tryptophan metabolism in human fibroblasts by transforming growth factor-beta: selective inhibition of indoleamine 2,3-dioxygenase and tryptophanyl-tRNA synthetase gene expression.

Alterations in the rate of cellular tryptophan metabolism are involved in mediating important biological activities associated with cytokines and growth factors. Indoleamine 2,3-dioxygenase (IDO) and tryptophanyl-tRNA synthetase are enzymes of tryptophan metabolism whose expression in a variety of cells and tissues is highly inducible by interferon-gamma (IFN-gamma). Transforming growth factor-beta (TGF-beta) antagonizes many cellular responses to IFN-gamma. The interaction of these two cytokines plays an important role in maintaining homeostasis during inflammation and repair. In human skin and synovial fibroblasts in vitro, TGF-beta caused time- and dose-dependent abrogation of IFN-gamma-stimulated expression of IDO and tryptophanyl-tRNA synthetase mRNAs. The inhibition was selective and did not appear to be due to down-regulation of IFN-gamma signaling by TGF-beta. In parallel with its effect on IDO mRNA expression, TGF-beta caused a marked reduction in intracellular IDO protein levels and abrogated IDO activity and tryptophan catabolism in these cells induced by IFN-gamma. IFN-gamma caused a rapid and striking increase in the amount of IDO heterogeneous nuclear pre-mRNA and induced transcription of the IDO gene, as demonstrated by transient transfection assays. TGF-beta partially reversed this stimulation. IFN regulatory factor (IRF)-1 and stat1 are cellular intermediates in IFN signaling. Both are implicated in activation of IDO transcription in response to IFN-gamma. The stimulation by IFN-gamma of IRF-1 protein and mRNA expression was not prevented by treatment of fibroblasts with TGF-beta. Furthermore, gel mobility shift assays indicated that TGF-beta did not inhibit the induction of stat1 and IRF-1 binding activity to their cognate DNA recognition sites in the IDO gene promoter. In contrast, the stability of IDO mRNA transcripts was reduced in fibroblasts treated with TGF-beta, as shown by determination of mRNA half-lives following blockade of transcription with 5,6-dichlorobenzimidazole riboside. The findings indicate that TGF-beta prevents the induction of IDO and tryptophanyl-tRNA synthetase gene expression in fibroblasts. The repression of IDO expression by TGF-beta is mediated at both transcriptional and posttranscriptional levels. These results implicate TGF-beta in the negative regulation of tryptophan metabolism, provide evidence for the molecular basis of this regulation, and indicate that cellular tryptophan metabolism is under tight immunological control.

Adult↗
PubMed2006

Relaxed transcription in Arabidopsis mitochondria is counterbalanced by RNA stability control mediated by polyadenylation and polynucleotide phosphorylase.

Plant mitochondrial genomes are extraordinarily large and complex compared to their animal counterparts, due to the presence of large noncoding regions. Multiple promoters are common for plant mitochondrial genes, and transcription exhibits little or no modulation. Mature functional RNAs are produced through various posttranscriptional processes, and control of RNA stability has a major impact on RNA abundance. This control involves polyadenylation which targets RNA for degradation by polynucleotide phosphorylase (PNPase). Here, we have analyzed polyadenylated RNA fragments from Arabidopsis plants down-regulated for PNPase (PNP- plants). Because of their polyadenylated status and the accumulation of the corresponding RNA in PNP- versus wild-type plants, these sequences represent mitochondrial RNA degradation tags. Analysis of these tags revealed that PNPase is involved in degrading rRNA and tRNA maturation by-products but also RNA transcribed from regions that are in some cases highly expressed although lacking known functional genes. Some of these transcripts, such as RNA containing chimeric open reading frames created by recombination or antisense RNA transcribed on the opposite strand of a known gene, may present potential detrimental effects to mitochondrial function. Taken together, our data show that the relaxed transcription in Arabidopsis mitochondria is counterbalanced by RNA stability control mediated by polyadenylation and PNPase.

Arabidopsis↗
PubMed1986

Analysis of RNA polymerase III transcription complexes by gel filtration.

We studied the in vitro assembly and stability of RNA polymerase III transcription complexes on the 5 S RNA and tRNAMet genes of Xenopus and the VA genes of adenovirus. Complete and partial assemblies were formed on these genes using transcription factor IIIA from Xenopus ovaries and factors IIIB and IIIC from HeLa cells. The complexes were purified away from unbound factors by filtration through Sepharose 4B columns and then assayed for transcription in the presence of Xenopus polymerase III. The 5 S gene complexes were also investigated using a postlabeling DNase I footprinting technique that we devised. The binding of factor IIIA to the 5 S gene facilitated the binding of factor IIIC; this subassembly was required for factor IIIB to bind. On the VA I and tRNA genes, factor IIIC alone bound and allowed IIIB to bind. RNA polymerase bound last to form a preinitiation complex, but it was less stably affixed than any of the factors. The complete factor complexes on the 5 S and VA I genes were strikingly stable to brief exposure to high salt concentrations, and the stability of the factor IIIB interaction was limiting. Two modes of IIIC binding were distinguished that differed in stability and specificity. Assembly of the complexes did not require ATP, and faithful transcription occurred when adenyl-5'-yl imidodiphosphate was substituted for ATP.

Adenosine Triphosphate↗
PubMed1989

Time of action of 4.5 S RNA in Escherichia coli translation.

A new class of suppressor mutants helps to define the role of 4.5 S RNA in translation. The suppressors reduce the requirement for 4.5 S RNA by increasing the intracellular concentration of uncharged tRNA. Suppression probably occurs by prolonging the period in which translating ribosomes have translocated but not yet released the uncharged tRNA, indicating that this is the point at which 4.5 S RNA enters translation. The release of 4.5 S RNA from polysomes is affected by antibiotics that inhibit protein synthesis. The antibiotic-sensitivity of this release indicates that 4.5 S RNA exits the ribosome following translocation and prior to release of protein synthesis elongation factor G. These results indicate that 4.5 S RNA acts immediately after ribosomal translocation. A model is proposed in which 4.5 S RNA stabilizes the post-translocation state by replacing 23 S ribosomal RNA as a binding site for elongation factor G. The 4.5 S RNA-requirement of mutants altered in 23 S ribosomal RNA support this model.

Anti-Bacterial Agents↗
PubMed1978

Synthesis and activities of branched-chain aminoacyl-tRNA synthetases in threonine deaminase mutants of Escherichia coli.

Valyl-, isoleucyl-, and leucyl-tRNA synthetase activities were examined in an Escherichia coli K-12 strain that possessed a deletion of three genes of the ilv gene cluster, ilvD, A, and C, and in a strain with the same deletion that also carried the lambdadilvCB bacteriophage. It was observed that the branched-chain tRNA synthetase activities of both strains were considerably less than those of the normal strain during growth in unrestricted medium. Furthermore, during an isoleucine limitation, there was a further reduction in isoleucyl-tRNA synthetase activity and an absence of the isoleucine-mediated derepression of valyl-tRNA synthetase formation in both of these mutants, as compared with the normal strain. In addition, it was observed that these branched-chain synthetase activities were reduced in steady-state cultures of several ilvA point mutants. However, upon the introduction of the ilv operon to these ilvA mutants by use of lambda bacteriophage, there was a specific increase in the branched-chain synthetase activities to levels comparable to those of the normal strain. These results support our previous findings that the stability and repression control of synthesis of these synthetases require some product(s) missing in the ilvDAC deletion strain and strongly suggest this component is some form of the ilvA gene product, threonine deaminase.

Amino Acyl-tRNA Synthetases↗
PubMed2002

Mammalian translation initiation factor eIF1 functions with eIF1A and eIF3 in the formation of a stable 40 S preinitiation complex.

We have examined the role of the mammalian initiation factor eIF1 in the formation of the 40 S preinitiation complex using in vitro binding of initiator Met-tRNA (as Met-tRNA(i).eIF2.GTP ternary complex) to 40 S ribosomal subunits in the absence of mRNA. We observed that, although both eIF1A and eIF3 are essential to generate a stable 40 S preinitiation complex, quantitative binding of the ternary complex to 40 S subunits also required eIF1. The 40 S preinitiation complex contained, in addition to eIF3, both eIF1 and eIF1A in a 1:1 stoichiometry with respect to the bound Met-tRNA(i). These three initiation factors also bind to free 40 S subunits, and the resulting complex can act as an acceptor of the ternary complex to form the 40 S preinitiation complex (40 S.eIF3.eIF1.eIF1A.Met-tRNA(i).eIF2.GTP). The stable association of eIF1 with 40 S subunits required the presence of eIF3. In contrast, the binding of eIF1A to free 40 S ribosomes as well as to the 40 S preinitiation complex was stabilized by the presence of both eIF1 and eIF3. These studies suggest that it is possible for eIF1 and eIF1A to bind the 40 S preinitiation complex prior to mRNA binding.

Animals↗
PubMed2026

Genomic characterisation and lytic potential of phage SF01 against multidrug-resistant Salmonella enterica subsp. enterica, a key agent of infection in poultry.

1. Salmonella enterica remains the key cause of salmonellosis in poultry, causing high morbidity and mortality. Due to the unprecedented resistance of S. enterica to antibiotics, bacteriophages have emerged as a powerful alternative to conventional antibiotics treatment for salmonellosis.2. In this study, a strain was isolated from infected broiler chickens and whole-genome sequencing (WGS) identified the strain SFD-01 as S. enterica subsp. enterica. Bioinformatics analyses revealed that the genome was 4.6 Mb in size with 4559 coding sequences (CDS), 77 tRNAs and 4 rRNAs. Additionally, 119 virulence genes, 125 antimicrobial resistance genes, 5 mobile genetic elements, 2 prophages and multiple copies of pathogenicity islands (SPI) were identified in the genome.3. To address this, bacteriophage SF01 was isolated from wastewater near a chicken slaughterhouse against host strain SFD-01. Transmission electron microscopy revealed that the phage had an 85-nm icosahedral head and a 130-nm long contractile tail. The Felixounavirus SF01 exhibited high stability across pH 3-9. Phage lytic activity at a multiplicity of infection of 0.01 restricted the bacterial growth.4. Whole genome analysis (WGS) identified phage SF01 as a Felixounavirus with 88-kb genome composed of 174 CDS, 20 tRNA genes and with no lysogenic markers, resistance genes or virulence factors. The strict lytic potential of phage SF01 makes it a highly viable option for use in the potential biocontrol of the novel strain S. enterica subsp. enterica serotype 42:z4,z23.

Felixounavirus↗
PubMed2002

Structure of small protein B: the protein component of the tmRNA-SmpB system for ribosome rescue.

Small protein B (SmpB) is an essential component of the highly conserved tmRNA-SmpB system that has the dual function of releasing stalled ribosomes from damaged messenger RNAs and targeting incompletely synthesized protein fragments for degradation. Nuclear magnetic resonance (NMR) analysis of SmpB from Aquifex aeolicus revealed an antiparallel beta-barrel structure, with three helices packed outside the core of the barrel. While the overall structure of SmpB appears to be unique, the structure does contain an embedded oligonucleotide binding fold; in this respect SmpB has similarity to several other RNA-binding proteins that are known to be associated with translation, including IF1, ribosomal protein S17 and the N-terminal domain of aspartyl tRNA synthetase. Conserved amino acids on the protein surface that are most likely to directly interact with the tmRNA were identified. The presence of widely separated clusters of conserved amino acids suggests that SmpB could function either by stabilizing two distal regions of the tmRNA, or by facilitating an interaction between the tmRNA and another component of the translational apparatus.

Amino Acid Sequence↗
PubMed1995

Partition of aminoacyl-tRNA synthetases in two different structural classes dating back to early metabolism: implications for the origin of the genetic code and the nature of protein sequences.

We describe, on the molecular level, a possible fuzzy and primordial translation apparatus capable of synthesizing polypeptides from nucleic acids in a world containing a mixture of coevolving molecules of RNA and proteins already arranged in metabolic cycles (including cofactors). Close attention is paid to template-free systems because they are believed to be the immediate ancestors of this primordial translation apparatus. The two classes of aminoacyl-tRNA synthetases (aaRSs), as seen today, are considered as the remnants of such a simple imprecise translation apparatus and are used as guidelines for the construction of the model. Earlier theoretical work by Bedian on a related system is invoked to show how specificity and stability could have been achieved automatically and rather quickly, starting from such an imprecise system, i.e., how the encoded synthesis of proteins could have appeared. Because of the binary nature of the underlying proto-code, the first genetically encoded proteins would then have been alternating copolymers with a high degree of degeneracy, but not random. Indeed, a clear signal for alternating hydrophobic and hydrophilic residues in present-day protein sequences can be detected. Later evolution of the genetic code would have proceeded along lines already discussed by Crick. However, in the initial stages, the translation apparatus proposed here is in fact very similar to the one postulated by Woese, only here it is given a molecular framework. This hypothesis departs from the paradigm of the RNA world in that it supposes that the origin of the genetic code occurred after the apparition of some functional (statistical) proteins first. Implications for protein design are also discussed.

Amino Acid Sequence↗
PubMed1989

Transcription factor IIIB generates extended DNA interactions in RNA polymerase III transcription complexes on tRNA genes.

Transcription complexes that assemble on tRNA genes in a crude Saccharomyces cerevisiae cell extract extend over the entire transcription unit and approximately 40 base pairs of contiguous 5'-flanking DNA. We show here that the interaction with 5'-flanking DNA is due to a protein that copurifies with transcription factor TFIIIB through several steps of purification and shares characteristic properties that are normally ascribed to TFIIIB: dependence on prior binding of TFIIIC and great stability once the TFIIIC-TFIIIB-DNA complex is formed. SUP4 gene (tRNATyr) DNA that was cut within the 5'-flanking sequence (either 31 or 28 base pairs upstream of the transcriptional start site) was no longer able to stably incorporate TFIIIB into a transcription complex. The TFIIIB-dependent 5'-flanking DNA protein interaction was predominantly not sequence specific. The extension of the transcription complex into this DNA segment does suggest two possible explanations for highly diverse effects of flanking-sequence substitutions on tRNA gene transcription: either (i) proteins that are capable of binding to these upstream DNA segments are also potentially capable of stimulating or interfering with the incorporation of TFIIIB into transcription complexes or (ii) 5'-flanking sequence influences the rate of assembly of TFIIIB into stable transcription complexes.

Base Sequence↗
PubMed1991

Studies on the interaction of T7 RNA polymerase with a DNA template containing a site-specifically placed psoralen cross-link. II. Stability and some properties of elongation complexes.

We constructed a 66 base-pair DNA template capable of supporting transcription by T7 RNA polymerase. This template had a psoralen cross-link downstream from a T7 promoter such that a 36 (+1) nucleotide transcript was synthesized at the time the T7 polymerase came to a stop at the cross-link. The stability of elongation complexes formed on this template, and the effect of different factors that are known to affect polymerase-DNA interactions was investigated by non-denaturing gel electrophoresis and gel filtration chromatography. We found that an elongation complex could lose its RNA component but the T7 polymerase still remained attached to the DNA template for extended periods of time (at least up to 18 h). This type of an elongation complex, bereft of its nascent RNA transcript, is called a quasi-elongation complex. DNase I footprinting within gel slices indicated that the polymerase molecules were arrested at the psoralen cross-link on the DNA template in the quasi-elongation complexes. The quasi-elongation complexes were found to be extremely stable in 0.5 M-NaCl and in 0.2 M-NaCl plus 60 mM-MgCl2, and could withstand temperatures up to 42 degrees C. The quasi-elongation complexes were destabilized by heparin and excess calf thymus DNA. Excess tRNA caused only a minimal degree of disruption. Non-promoter-containing plasmid DNAs did not have a destabilizing effect on the quasi-elongation complexes. Interestingly, it was observed that in a T7 ternary transcriptional complex arrested by a psoralen cross-link, the nascent RNA transcript could be stabilized from release by the presence in trans of a plasmid DNA bearing a T7 promoter and a T7 terminator. Such a stabilization against RNA release was not observed with plasmid DNAs containing either only a promoter or a terminator. The elongation complexes were stable during gel filtration through Sephacryl S-300 HR. However, it was found that 30% to 45% of the labeled RNA was retained during gel filtration as RNA that was apparently free from ternary complexes.

Base Sequence↗
PubMed2004

On the role of the starved codon and the takeoff site in ribosome bypassing in Escherichia coli.

Translating ribosomes can skip over stretches of messenger RNA and resume protein chain elongation after a "bypassed" region. We have previously shown that limitation for isoleucyl-tRNA can initiate a ribosome bypass when an AUA codon is in the ribosomal A-site. We have now generalized this effect to other "hungry" codons calling for four different limiting aminoacyl-tRNA species, suggesting that a pause at any A-site will have this effect. We have assessed bypassing in a large family of reporters with nearly every different triplet in the "takeoff site", i.e. the P-site on the 5' side of the hungry codon, and an identical "landing site" codon 16 nucleotides downstream. The different takeoff sites vary over a factor of 50 in bypassing proficiency. At least part of this variation appears to reflect stability of the codon Colon, two colons anticodon interaction at the takeoff site, as indicated by the following: (a) the bypassing proficiency of different tRNAs shows a rough correlation with the frequency of A Colon, two colons U as opposed to G Colon, two colons C pairs in the codon Colon, two colons anticodon association; (b) specific tRNAs bypass more frequently from codons ending in U than from their synonym ending in C; (c) an arginine tRNA with Inosine in the wobble position which reads CGU, CGC, and CGA bypasses much more frequently from the last codon than the first two synonyms.

Amino Acid Sequence↗
PubMed1994

The active site of yeast aspartyl-tRNA synthetase: structural and functional aspects of the aminoacylation reaction.

The crystal structures of the various complexes formed by yeast aspartyl-tRNA synthetase (AspRS) and its substrates provide snapshots of the active site corresponding to different steps of the aminoacylation reaction. Native crystals of the binary complex tRNA-AspRS were soaked in solutions containing the two other substrates, ATP (or its analog AMPPcP) and aspartic acid. When all substrates are present in the crystal, this leads to the formation of the aspartyl-adenylate and/or the aspartyl-tRNA. A class II-specific pathway for the aminoacylation reaction is proposed which explains the known functional differences between the two classes while preserving a common framework. Extended signature sequences characteristic of class II aaRS (motifs 2 and 3) constitute the basic functional unit. The ATP molecule adopts a bent conformation, stabilized by the invariant Arg531 of motif 3 and a magnesium ion coordinated to the pyrophosphate group and to two class-invariant acidic residues. The aspartic acid substrate is positioned by a class II invariant acidic residue, Asp342, interacting with the amino group and by amino acids conserved in the aspartyl synthetase family. The amino acids in contact with the substrates have been probed by site-directed mutagenesis for their functional implication.

Acylation↗