SEARCH · PubMed Health

Results for “RNA, Transfer, Met”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 109 records · Page 6Linked to original sources

PubMed2005

Inhibition of HIV-1 gene expression by retroviral vector-mediated small-guide RNAs that direct specific RNA cleavage by tRNase ZL.

The tRNA 3'-processing endoribonuclease (tRNase Z or 3' tRNase; EC 3.1.26.11) is an essential enzyme that removes the 3' trailer from pre-tRNA. The long form (tRNase ZL) can cleave a target RNA in vitro at the site directed by an appropriate small-guide RNA (sgRNA). Here, we investigated whether this sgRNA/tRNase ZL strategy could be applied to gene therapy for AIDS. We tested the ability of four sgRNA-expression plasmids to inhibit HIV-1 gene expression in COS cells, using a transient-expression assay. The three sgRNAs guide inhibition of HIV-1 gene expression in cultured COS cells. Analysis of the HIV-1 mRNA levels suggested that sgRNA directed the tRNase ZL to mediate the degradation of target RNA. The observation that sgRNA was localized primarily in nuclei suggests that tRNase ZL cleaves the HIV-1 mRNA when complexed with sgRNA in this location. We also examined the ability of two retroviral vectors expressing sgRNA to suppress HIV-1 expression in HIV-1-infected Jurkat T cells. sgRNA-SL4 suppressed HIV-1 expression almost completely in infected cells for up to 18 days. These results suggest that the sgRNA/tRNase ZL approach is effective in downregulating HIV-1 gene expression.

Animals↗

PubMed2006

Translation initiation by using various N-acylaminoacyl tRNAs.

Bioactive peptides isolated from natural sources have diverse acyl groups on the N-terminus. It is difficult to synthesize these peptides in vitro translation system because ribosomal peptide synthesis generally limits the N-terminal group to be N-formylmethionine (fMet). To overcome this restriction, we developed a novel methodology for the ribosomal synthesis of peptides having various terminal N-acyl groups with desired amino acids. In this methodology, two technologies, Flexizyme system consisting of artificial ribozymes and a reconstitute E. coli cell-free translation system (PURE system), were used. First, an amino acid carrying a desired N-acyl group was charged onto an initiation tRNA by the Flexizyme system. The addition of this N-acyl-aminoacyl-tRNA (N-acyl-aa-tRNA) to the PURE system allowed us to initiate the peptide synthesis with the designated N-acyl-amino acid. By means of this methodology, the translation was exclusively initiated by various N-terminal acyl groups as well as amino acids without contamination of N-formylmethionine.

Aminoacylation↗

PubMed2003

Translation elongation after assembly of ribosomes on the Cricket paralysis virus internal ribosomal entry site without initiation factors or initiator tRNA.

Reconstitution of translation elongation from purified components confirmed that ribosomes that assembled on the Cricket paralysis virus intercistronic internal ribosomal entry site (IRES) without the involvement of initiation factors or initiator tRNA were active in elongation and are, therefore, true initiation complexes. The first elongation cycle occurred without peptide bond formation on 80S ribosomes that did not contain tRNA in the P site. It required elongation factors 1A and 2 and A site-cognate aminoacylated tRNA. Cycloheximide arrested ribosomes on the IRES only after two cycles of elongation, when the first deacylated tRNA reached the E-site after translocation from the A-site.

Amino Acid Sequence↗

PubMed1990

Use of natural mRNAs in the cell-free protein-synthesizing systems of the moderate halophile Vibrio costicola.

In vitro protein synthesis was studied in extracts of the moderate halophile Vibrio costicola by using as mRNAs the endogenous mRNA of V. costicola and the RNA of the R17 bacteriophage of Escherichia coli. Protein synthesis (amino acid incorporation) was dependent on the messenger, ribosomes, soluble cytoplasmic factors, energy source, and tRNA(FMet) (in the R17 RNA system) and was inhibited by certain antibiotics. These properties indicated de novo protein synthesis. In the V. costicola system directed by R17 RNA, a protein of the same electrophoretic mobility as the major coat protein of the R17 phage was synthesized. Antibiotic action and the response to added tRNA(FMet) showed that protein synthesis in the R17 RNA system, but not in the endogenous messenger system, absolutely depended on initiation. Optimal activity of both systems was observed in 250 to 300 mM NH4+ (as glutamate). Higher salt concentrations, especially those with Cl- as anion, were generally inhibitory. The R17 RNA-directed system was more sensitive to Cl- ions than the endogenous system was. Glycine betaine stimulated both systems and partly overcame the toxic effects of Cl- ions. Both systems required Mg2+, but in lower concentrations than the polyuridylic acid-directed system previously studied. Initiation factors were removed from ribosomes by washing with 3.0 to 3.5 M NH4Cl, concentrations about three times as high as that needed to remove initiation factors from E. coli ribosomes. Washing with 4.0 M NH4Cl damaged V. costicola ribosomes, although the initiation factors still functioned. Cl- ions inhibited the attachment of initiation factors to tRNA(FMet) but had little effect on binding of initiation factors to R17 RNA.

Bacterial Proteins↗

PubMed1995

Genetic instability of a MoMLV-based antisense double-copy retroviral vector designed for HIV-1 gene therapy.

We constructed a retroviral vector encoding a mutant tRNA(imet) gene followed by a HIV-1 rev-specific antisense sequence in the U3 region of the 3' long terminal repeat (LTR). This Moloney murine leukemia virus (MoMLV)-based double-copy retroviral vector was used to transduce human lymphoblastoid T-cell lines (CEM, Jurkat). In some clonal cell lines the expected short transcript initiated either from the 5' or 3' LTR tRNA-alpha rev gene was not detectable by Northern blot analyses of transduced, G418-resistant cells with an alpha rev-specific oligonucleotide probe. In other clonal cells, neither the short polymerase III transcript nor the full-length genomic polymerase II transcript (containing the 3' LTR tRNA-alpha rev gene) was detectable when compared with the transduced cell pool. Southern blot and DNA-polymerase chain reaction (PCR) analyses specific for the tRNA-alpha rev cassette in the 5' or 3' LTR of the retroviral vector suggested that the transfer of the 3' LTR U3 region to the 5' LTR was incorrect in most proviruses. These data were confirmed by DNA sequence analyses of several clonal lines demonstrating deletions and insertions. In summary, our results indicate that this retroviral vector design with direct repeats flanking the polymerase III transcription unit plus the alpha rev insert is prone to genetic rearrangements and consequently not useful for the development of gene therapy protocols.

Animals↗

PubMed1996

The A1 x U72 base pair conserved in eukaryotic initiator tRNAs is important specifically for binding to the eukaryotic translation initiation factor eIF2.

The formation of a specific ternary complex between eukaryotic initiation factor 2 (eIF2), the initiator methionyl-tRNA (Met-tRNA), and GTP is a critical step in translation initiation in the cytoplasmic protein-synthesizing system of eukaryotes. We show that the A1 x U72 base pair conserved at the end of the acceptor stem in eukaryotic and archaebacterial initiator methionine tRNAs plays an important role in this interaction. We changed the A1 x U72 base pair of the human initiator tRNA to G1 x C72 and expressed the wild-type and mutant tRNA genes in the yeast Saccharomyces cerevisiae by using constructs previously developed in our laboratory for expression of the human initiator tRNA gene in yeasts. We show that both the wild-type and mutant human initiator tRNAs are aminoacylated well in vivo. We have isolated the wild-type and mutant human initiator tRNAs in substantially pure form, free of the yeast initiator tRNA, and have analyzed their properties in vitro. The G1 x C72 mutation affects specifically the binding affinity of eIF2 for the initiator tRNA. It has no effect on the subsequent formation of 40S or 80S ribosome initiator Met-tRNA-AUG initiation complexes in vitro or on the puromycin reactivity of the Met-tRNA in the 80S initiation complex.

Base Sequence↗

PubMed2001

The appended C-domain of human methionyl-tRNA synthetase has a tRNA-sequestering function.

An ancillary RNA-binding domain is appended to the C-terminus of human methionyl-tRNA synthetase. It comprises a helix-turn-helix (HTH) motif related to the repeated units of the linker region of bifunctional glutamyl-prolyl-tRNA synthetase, and a specific C-terminal KGKKKK lysine-rich cluster (LRC). Here we show by gel retardation and tRNA aminoacylation experiments that these two regions are important for tRNA binding. However, the two pieces of this bipartite RNA-binding domain are functionally distinct. Analysis of MetRS mutant enzymes revealed that the HTH motif is more specifically endowed with a tRNA-sequestering activity and confers on MetRS a rate-limiting dissociation of aminoacylated tRNA. Elongation factor EF-1alpha enhanced the turnover in the aminoacylation reaction. In contrast, the LRC region is most probably involved in accelerating the association step of deacylated tRNA. These two nonredundant RNA-binding motifs strengthen tRNA binding by the synthetase. The native form of MetRS, containing the C-terminal RNA-binding domain, behaves as a processive enzyme; release of the reaction product is not spontaneous, but may be synchronized with the subsequent step of the tRNA cycle through EF-1alpha-assisted dissociation of Met-tRNA(Met). Therefore, the eukaryotic-specific C-domain of human MetRS may have a dual function. It may ensure an efficient capture of tRNA(Met) under conditions of suboptimal deacylated tRNA concentration prevailing in vivo, and may instigate direct transfer of aminoacylated tRNA from the synthetase to elongation factor EF-1alpha.

Amino Acid Sequence↗

PubMed1994

Characterization of a cell division gene from Bacillus subtilis that is required for vegetative and sporulation septum formation.

We report the cloning and characterization of a cell division gene, herein designated divIC, from the gram-positive, spore-forming bacterium Bacillus subtilis. This gene was previously identified on the basis of a temperature-sensitive mutation, div-355, that blocks septum formation at restrictive temperatures. We show that the divIC gene is a 125-codon open reading frame that is capable of encoding a protein of 14.7 kDa and that div-355 is a 5-bp duplication near the 3' end of the open reading frame. We also show that divIC is an essential gene by use of an in vitro-constructed null mutation. In confirmation and extension of earlier results, we show that divIC is necessary for both vegetative and sporulation septum formation, and we demonstrate that it is required for the activation of genes expressed under the control of the sporulation transcription factors sigma F and sigma E. The divIC gene is located 1.3 kb upstream of the coding sequence for the sporulation gene spoIIE. Between divIC and spoIIE is a 128-codon open reading frame whose predicted product contains a region of similarity to the RNA-binding domains of polynucleotide phosphorylase and ribosomal protein S1 from Escherichia coli and two putative tRNA genes for methionyl-tRNA and glutamyl-tRNA, the gene order being divIC orf128 tRNA(Met) tRNA(Glu) spoIIE.

Amino Acid Sequence↗

PubMed1996

Ligand interactions with eukaryotic translation initiation factor 2: role of the gamma-subunit.

Eukaryotic translation initiation factor 2 (eIF-2) comprises three non-identical subunits alpha, beta and gamma. In vitro, eIF-2 binds the initiator methionyl-tRNA in a GTP-dependent fashion. Based on similarities between eukaryotic eIF-2gamma proteins and eubacterial EF-Tu proteins, we previously proposed a major role for the gamma-subunit in binding guanine nucleotide and tRNA. We have tested this hypothesis by examining the biochemical activities of yeast eIF-2 purified from wild-type strains and strains harboring mutations in the eIF-2gamma structural gene (GCD11) predicted to alter ligand binding by eIF-2. The alteration of tyrosine 142 in yeast eIF-2gamma, corresponding to histidine 66 in Escherichia coli EF-Tu, dramatically reduced the affinity of eIF-2 for Met-tRNAi(Met) without affecting the k(off) value for guanine nucleotides. In contrast, non-lethal substitutions at a conserved lysine residue (K250) in the putative guanine ring-binding loop increased the off-rate for GDP, thereby mimicking the function of the guanine nucleotide exchange factor eIF-2B, without altering the apparent dissociation constant for Met-tRNAi(Met). For eIF-2[gamma-K250R], the increased off-rate also seen for GTP was masked by the presence of Met-tRNAi(Met) in vitro. In vivo, increasing the dose of the yeast initiator tRNA gene suppressed the slow-growth phenotype and reduced GCN4 expression in gcd11-K250R and gcd11-Y142H strains. These studies indicate that the gamma-subunit of eIF-2 does indeed provide EF-Tu-like function to the eIF-2 complex, and further suggest that the level of Met-tRNAi(Met) is critical for maintaining wild-type rates of initiation in vivo.

Electrophoresis, Polyacrylamide Gel↗

PubMed1990

The role of mammalian initiation factor eIF-4D and its hypusine modification in translation.

Initiation factor eIF-4D functions late in the initiation pathway, apparently during formation of the first peptide bond. The factor is post-translationally modified at a specific lysine residue by reaction with spermidine and subsequent hydroxylation to form hypusine. A precursor form lacking hypusine is inactive in the assay for methionyl-puromycin synthesis, but activity is restored following in vitro modification to deoxyhypusine, thereby suggesting that the modification is essential for function. Since formylated methionyl-tRNA is less dependent on eIF-4D in the puromycin assay, we postulate that eIF-4D and its hypusine modification may stabilize charged Met-tRNA binding to the peptidyl transferase center of the 60S ribosomal subunit. Analysis of eIF-4D genes in yeast indicate that eIF-4D and its hypusine modification are essential for cell growth.

HeLa Cells↗

PubMed2006

Distinct induction patterns and functions of two closely related interferon-inducible human genes, ISG54 and ISG56.

Human P54 and P56 proteins are tetratricopeptide proteins that are encoded by two closely related genes, ISG54 and ISG56. These genes are induced strongly but transiently when cells are treated with interferons or double-stranded RNA or infected with a variety of viruses. We observed that, although double-stranded RNA or Sendai virus infection induced the two genes with similar kinetics, their induction kinetics in response to interferon-beta were quite different. The induction kinetics by virus infection were also different between two cell lines. Functionally the two proteins were similar. Like P56, P54 bound to the translation initiation factor eIF3 and inhibited translation. However, unlike P56, P54 bound to both the "e" and the "c" subunits of eIF3. Consequently, P54 inhibited two functions of eIF3. Like P56, it inhibited the ability of eIF3 to stabilize the eIF2 x GTP x Met-tRNA(i) ternary complex. But in addition, it also inhibited the formation of the 48 S pre-initiation complex between the 40 S ribosomal subunit and the 20 S complex composed of eIF3, ternary complex, eIF4F, and mRNA. Thus, although similar in structure, the human P54 and P56 proteins are induced differently and function differently.

Adaptor Proteins, Signal Transducing↗

PubMed2000

The Gcd10p/Gcd14p complex is the essential two-subunit tRNA(1-methyladenosine) methyltransferase of Saccharomyces cerevisiae.

The modified nucleoside 1-methyladenosine (m(1)A) is found at position 58 in the TPsiC loop of many eukaryotic tRNAs. The absence of m(1)A from all tRNAs in Saccharomyces cerevisiae mutants lacking Gcd10p elicits severe defects in processing and stability of initiator methionine tRNA (tRNA(i)(Met)). Gcd10p is found in a complex with Gcd14p, which contains conserved motifs for binding S-adenosylmethionine (AdoMet). These facts, plus our demonstration that gcd14Delta cells lacked m(1)A, strongly suggested that Gcd10p/Gcd14p complex is the yeast tRNA(m(1)A)methyltransferase [(m(1)A)MTase]. Supporting this prediction, affinity-purified Gcd10p/Gcd14p complexes used AdoMet as a methyl donor to synthesize m(1)A in either total tRNA or purified tRNA(i)(Met) lacking only this modification. Kinetic analysis of the purified complex revealed K(M) values for AdoMet or tRNA(i)(Met) of 5.0 microM and 2.5 nM, respectively. Mutations in the predicted AdoMet-binding domain destroyed GCD14 function in vivo and (m(1)A)MTase activity in vitro. Purified Flag-tagged Gcd14p alone had no enzymatic activity and was severely impaired for tRNA-binding compared with the wild-type complex, suggesting that Gcd10p is required for tight binding of the tRNA substrate. Our results provide a demonstration of a two-component tRNA MTase and suggest that binding of AdoMet and tRNA substrates depends on different subunits of the complex.

Binding Sites↗

PubMed2002

Endogenous expression of a high-affinity pseudoknot RNA aptamer suppresses replication of HIV-1.

Aptamers, small oligonucleotides derived from an in vitro evolution process called SELEX, are promising therapeutic and diagnostic agents. Although very effective in vitro, only a few examples are available showing their potential in vivo. We have analyzed the effect of a well characterized pseudoknot RNA aptamer selected for tight binding to human immunodeficiency virus (HIV) type 1 reverse transcriptase on HIV replication. Transient intracellular expression of a chimeric RNA consisting of the human initiator tRNA(Met) (tRNA(Meti))/aptamer sequence in human 293T cells showed inhibition of HIV particle release by >75% when the cells were co-transfected with proviral HIV-1 DNA. Subsequent virus production of human T-lymphoid C8166 cells, infected with viral particles derived from co-transfected 293T cells, was again reduced by >75% as compared with the control. As the observed effects are additive, in this model for virus spread, the total reduction of HIV particle formation by transient intracellular expression of the pseudoknot RNA aptamer amounts to >95%. Low-dose HIV infection of human T cells stably expressing the aptamer did not show any virus replication over a period of 35 days. This is the first example of an RNA aptamer selected against a viral enzyme target to show powerful antiviral activity in HIV-1-permissive human T-lymphoid cell lines.

Base Sequence↗

PubMed2000

Effective suppression of HIV-1 gene expression by a mammalian tRNA 3' processing endoribonuclease and external guide sequence oligozymes.

We examined the suppression of virus expression by cleavage of the HIV-1 RNA gene using a mammalian tRNA 3' processing endoribonuclease and an External Guide Sequence Oligozyme (EGS) in vivo. We constructed an EGS expression vector that used the tRNA(met) promoter as an expression cassette for EGS. The EGS expression vector was targeted to the upstream region of gag, region. The EGS expression vector was co-transfected into COS cells with the HIV-1 gene plasmid vector. As compared with the EGS non-expressing cells and the EGS expressing cells, the EGS expressing cells with the targeted gag start codon had a clearly decreased amount of the HIV-1 gag p24 protein. The EGS expressing cells with the targeted gag start codon showed effective suppression of HIV-1 gene expression. Thus, these studies describe novel gene targeting agents for the inhibition of gene expression and antiviral activity.

Animals↗

PubMed2000

Initiation factor 3-induced structural changes in the 30 S ribosomal subunit and in complexes containing tRNA(f)(Met) and mRNA.

Initiation factor 3 (IF3) acts to switch the decoding preference of the small ribosomal subunit from elongator to initiator tRNA. The effects of IF3 on the 30 S ribosomal subunit and on the 30 S.mRNA. tRNA(f)(Met) complex were determined by UV-induced RNA crosslinking. Three intramolecular crosslinks in the 16 S rRNA (of the 14 that were monitored by gel electrophoresis) are affected by IF3. These are the crosslinks between C1402 and C1501 within the decoding region, between C967xC1400 joining the end loop of a helix of 16 S rRNA domain III and the decoding region, and between U793 and G1517 joining the 790 end loop of 16 S rRNA domain II and the end loop of the terminal helix. These changes occur even in the 30 S.IF3 complex, indicating they are not mediated through tRNA(f)(Met) or mRNA. UV-induced crosslinks occur between 16 S rRNA position C1400 and tRNA(f)(Met) position U34, in tRNA(f)(Met) the nucleotide adjacent to the 5' anticodon nucleotide, and between 16 S rRNA position C1397 and the mRNA at positions +9 and +10 (where A of the initiator AUG codon is +1). The presence of IF3 reduces both of these crosslinks by twofold and fourfold, respectively. The binding site for IF3 involves the 790 region, some other parts of the 16 S rRNA domain II and the terminal stem/loop region. These are located in the front bottom part of the platform structure in the 30 S subunit, a short distance from the decoding region. The changes that occur in the decoding region, even in the absence of mRNA and tRNA, may be induced by IF3 from a short distance or could be caused by the second IF3 structural domain.

Alkalies↗

PubMed2002

Evidence that ternary complex (eIF2-GTP-tRNA(i)(Met))-deficient preinitiation complexes are core constituents of mammalian stress granules.

Environmental stress-induced phosphorylation of eIF2alpha inhibits protein translation by reducing the availability of eIF2-GTP-tRNA(i)Met, the ternary complex that joins initiator tRNA(Met) to the 43S preinitiation complex. The resulting untranslated mRNA is dynamically routed to discrete cytoplasmic foci known as stress granules (SGs), a process requiring the related RNA-binding proteins TIA-1 and TIAR. SGs appear to be in equilibrium with polysomes, but the nature of this relationship is obscure. We now show that most components of the 48S preinitiation complex (i.e., small, but not large, ribosomal subunits, eIF3, eIF4E, eIF4G) are coordinately recruited to SGs in arsenite-stressed cells. In contrast, eIF2 is not a component of newly assembled SGs. Cells expressing a phosphomimetic mutant (S51D) of eIF2alpha assemble SGs of similar composition, confirming that the recruitment of these factors is a direct consequence of blocked translational initiation and not due to other effects of arsenite. Surprisingly, phospho-eIF2alpha is recruited to SGs that are disassembling in cells recovering from arsenite-induced stress. We discuss these results in the context of a translational checkpoint model wherein TIA and eIF2 are functional antagonists of translational initiation, and in which lack of ternary complex drives SG assembly.

Animals↗

PubMed1994

The RNA-binding properties of protein synthesis initiation factor eIF-2.

Protein synthesis initiation factor eIF-2 bound ATP in the presence or absence of Mg2+ ions. ATP impaired the binding of GTP or GDP to eIF-2. However, excess GTP did not significantly decrease the binding of ATP to eIF-2, suggesting eIF-2 has distinct ATP and GTP binding sites. Highly purified eIF-2 can bind mRNA, and this did not require the mRNA to be capped. mRNA binding was saturable, and maximal binding corresponded to about 0.4 mol mRNA bound per mol eIF-2. GTP, and, at lower concentrations, GDP, inhibited the binding of mRNA to eIF-2. In addition, ATP and other nucleoside triphosphates decreased mRNA binding. The implications of these findings for the structure and function of eIF-2 are discussed. Preparations of eIF-2 deficient in the beta-subunit showed reduced ability to bind mRNA, suggesting that while it is not essential for mRNA binding, this subunit is involved in the interaction. Consistent with this is the observation that ultraviolet crosslinking of mRNA to eIF-2 resulted primarily in labelling of the beta-subunit. Subsequent analysis revealed that mRNA was cross-linked to the C-terminal region of eIF-2b which contains a putative Zn-finger structure.

Adenosine Triphosphate↗

PubMed2000

Interaction of fMet-tRNA(fMet) with the C-terminal domain of translational initiation factor IF2 from Bacillus stearothermophilus.

Analytical ultracentrifugation studies indicated that the C-terminal domains of IF2 comprising amino acid residues 520-741 (IF2 C) and 632-741 (IF2 C-2) bind fMet-tRNA with similar affinities (K(d) at 25 degrees C equal to 0.27 and 0.23 microM, respectively). Complex formation between fMet-tRNA(fMet) and IF2 C or IF2 C-2 is accompanied by barely detectable spectral changes as demonstrated by a comparison of the Raman spectra of the complexes with the calculated sum of the spectra of the individual components. These results and the temperature dependence of the K(d) of the protein-RNA complexes indicate that complex formation is not accompanied by obvious conformational changes of the components, and possibly depends on a rather small binding site comprising only a few interacting residues of both components.

Bacterial Proteins↗