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PubMed1996

Aminoacyl-tRNA recognition by the leucyl/phenylalanyl-tRNA-protein transferase.

We employ mutant and mischarged aminoacyl-tRNAs to characterize aminoacyl-tRNA recognition by the leucyl/phenylalanyl-tRNA-protein transferase (L/Ftransferase). Wild type Met-tRNAMetm (CAU anticodon) and mischarged Met-tRNAVal-1 (CAU anticodon) are substrates for the L/F-transferase during the NH2-terminal aminoacylation of alpha-casein, whereas Val-tRNAVal-1 (UAC), Val-tRNAMetm (UAC), and Arg-tRNAMetm (CCG, A20) are not. Mutations in the anticodon and extra arm of tRNALeu-1 do not measurably effect its ability to serve as a substrate for the L/F-transferase, and the dissociation constants of the complexes between L/F-transferase and either wild type Leu-tRNALeu-4 (UAA) or mutant Leu-tRNALeu-4 (CUA) are each 0.4 +/- 0.2 microM. The dissociation constants for the complexes between the L/F-transferase and uncharged tRNA, leucine methyl ester, and puromycin are all 10-1,000-fold greater than that of the Leu-tRNA.L/F-transferase complex. Dissociation of the Leu-tRNA.L/F-transferase complex is slow, relative to the rate calculated assuming that association is diffusion controlled. Finally, deoxyoligonucleotide.aminoacyl-tRNA hybrids (dO.AA-tRNAs) are employed to characterize the determinants of the Leu-tRNALeu-4 acceptor stem recognized by the L/F-transferase. A dO.AA-tRNA completely lacking acceptor stem base pairs remains a substrate for the L/F-transferase, whereas a dO.AA-tRNA containing a 2-base pair single-stranded region, at its 3' terminus, does not.

Acyltransferases↗

PubMed2001

Birth of a retroposon: the Twin SINE family from the vector mosquito Culex pipiens may have originated from a dimeric tRNA precursor.

SINEs are short interspersed repetitive elements found in many eukaryotic genomes and are believed to propagate by retroposition. Almost all SINEs reported to date have a composite structure made of a 5' tRNA-related region followed by a tRNA-unrelated region. Here, we describe a new type of tRNA-derived SINEs from the genome of the mosquito Culex pipiens. These elements, called TWINs, are approximately 220 bp long and reiterated at approximately 500 copies per haploid genome. TWINs have a unique structure compared with other tRNA-SINEs described so far. They consist of two tRNA(Arg)-related regions separated by a 39-bp spacer. Other tRNA-unrelated sequences include a 5-bp leader preceding the left tRNA-like unit and a short trailer located downstream of the right tRNA-like region. This 3' trailer is a 10-bp sequence that is ended by a TTTT motif and followed by a polyA tract of variable length. The right tRNA-like unit also contains a 16-bp sequence which is absent in the left one and appears to be located in the ancestral anticodon stem precisely at a position expected for a nuclear tRNA intron. According to this singular structure, we hypothesize that the TWIN: SINE family originated from an unprocessed polymerase III transcript containing two tRNA sequences. We suggest that some peculiar properties acquired by this dicistronic transcript, such as a polyA tail and a 3' stem-loop secondary structure, promote its retroposition by increasing its chances of being recognized by a reverse transcriptase encoded elsewhere in the C. pipiens genome.

Animals↗

PubMed1997

Mutation induction and mutation frequency decline in halogen light-irradiated Escherichia coli K-12 AB1157 strains.

The effects of halogen light irradiation on reversion of argE3-->Arg+ in E. coli K12 strain AB1157 and its mfd- mutant, and on mutation frequency decline (MFD) after transiently incubating irradiated bacteria under non-growing conditions were studied. The induction of mutations, the mutational specificity, and the MFD effect had the same characteristic features as those seen in E. coli B strains after irradiation with 254 nm UV light. MFD which is due to repair of premutagenic lesion in the transcribed strand of glnU gene and prevents mutations leading to supB formation, was not observed in halogen light-induced mutations in the mfd-1 strain. Overproduction of UmuD'C proteins led to a large increase in mutation frequency, which was much greater in mfd- than in mfd+ strains. In bacteria irradiated with halogen light and incubated immediately in a rich medium to express mutations, the formation of supB predominated strongly over that of supE(ochre) in mfd- cells but was at a similar level in mfd+ cells. Introduction of zcf117::Tn10 to AB1157 strain makes cells more sensitive to halogen light irradiation, whereas introduction of mfd-1 does not.

Arginine↗

PubMed2004

YIH1 is an actin-binding protein that inhibits protein kinase GCN2 and impairs general amino acid control when overexpressed.

The general amino acid control (GAAC) enables yeast cells to overcome amino acid deprivation by activation of the alpha subunit of translation initiation factor 2 (eIF2alpha) kinase GCN2 and consequent induction of GCN4, a transcriptional activator of amino acid biosynthetic genes. Binding of GCN2 to GCN1 is required for stimulation of GCN2 kinase activity by uncharged tRNA in starved cells. Here we show that YIH1, when overexpressed, dampens the GAAC response (Gcn- phenotype) by suppressing eIF2alpha phosphorylation by GCN2. The overexpressed YIH1 binds GCN1 and reduces GCN1-GCN2 complex formation, and, consistent with this, the Gcn- phenotype produced by YIH1 overexpression is suppressed by GCN2 overexpression. YIH1 interacts with the same GCN1 fragment that binds GCN2, and this YIH1-GCN1 interaction requires Arg-2259 in GCN1 in vitro and in full-length GCN1 in vivo, as found for GCN2-GCN1 interaction. However, deletion of YIH1 does not increase eIF2alpha phosphorylation or derepress the GAAC, suggesting that YIH1 at native levels is not a general inhibitor of GCN2 activity. We discovered that YIH1 normally resides in a complex with monomeric actin, rather than GCN1, and that a genetic reduction in actin levels decreases the GAAC response. This Gcn- phenotype was partially suppressed by deletion of YIH1, consistent with YIH1-mediated inhibition of GCN2 in actin-deficient cells. We suggest that YIH1 resides in a YIH1-actin complex and may be released for inhibition of GCN2 and stimulation of protein synthesis under specialized conditions or in a restricted cellular compartment in which YIH1 is displaced from monomeric actin.

Actins↗

PubMed2002

Nested evolution of a tRNA(Leu)(UAA) group I intron by both horizontal intron transfer and recombination of the entire tRNA locus.

The origin and evolution of bacterial introns are still controversial issues. Here we present data on the distribution and evolution of a recently discovered divergent tRNA(Leu)(UAA) intron. The intron shows a higher sequence affiliation with introns in tRNA(Ile)(CAU) and tRNA(Arg)(CCU) genes in alpha- and beta-proteobacteria, respectively, than with other cyanobacterial tRNA(Leu)(UAA) group I introns. The divergent tRNA(Leu)(UAA) intron is sporadically distributed both within the Nostoc and the Microcystis radiations. The complete tRNA gene, including flanking regions and intron from Microcystis aeruginosa strain NIVA-CYA 57, was sequenced in order to elucidate the evolutionary pattern of this intron. Phylogenetic reconstruction gave statistical evidence for different phylogenies for the intron and exon sequences, supporting an evolutionary model involving horizontal intron transfer. The distribution of the tRNA gene, its flanking regions, and the introns were addressed by Southern hybridization and PCR amplification. The tRNA gene, including the flanking regions, were absent in the intronless stains but present in the intron-containing strains. This suggests that the sporadic distribution of this intron within the Microcystis genus cannot be attributed to intron mobility but rather to an instability of the entire tRNA(Leu)(UAA) intron-containing genome region. Taken together, the complete data set for the evolution of this intron can best be explained by a model involving a nested evolution of the intron, i.e., wherein the intron has been transferred horizontally (probably through a single or a few events) to a tRNA(Leu)(UAA) gene which is located within a unstable genome region.

Base Sequence↗

PubMed1977

Yeast mitochondrial DNA specifies tRNA for 19 amino acids. Deletion mapping of the tRNA genes.

We have previously identified 14 aminoacyl tRNAs that are specified by yeast mitochondrial DNA (mtDNA). We now report four more amino acids (Arg, Cys, Trp, Thr) that acylate tRNAs which hybridize with mtDNA. Furthermore one of the two mitochondrial tRNAs that we had earlier demonstrated to be directly charged with glutamic acid responds to glutamine but not to glutamic acid codons. Thus Gln-tRNAGln appears to be formed by transamidation of a missense intermediate Glu-tRNAGln. This brings to 19 the number of amino acids which have corresponding tRNAs specified by mtDNA. Only tRNAAsn has not yet been shown to be a mtDNA transcript. We have also mapped the genes for the newly identified mitochondrial tRNAs, as well as several others that were previously identified but unmapped, by hybridization to the mtDNA of a series of petite deletion mutants. We now have ordered 20 mitochondrial tRNA genes (including two methionyl-tRNAs) wtih respect to the antibiotic resistance markers chloramphenicol (CR), erythromycin (ER), paromomycin (PR), and oligomycin I and II (ORI, ORII). Eighteen tRNA genes map between the C and E resistance markers. Only the serinyl-tRNA and glutamyl-tRNA genes are localized near the OI and OII resistance markers.

Amino Acyl-tRNA Synthetases↗

PubMed1993

Biochemical and functional characteristics of soluble MHC molecules derived from H-2Ld/Q10d chimeric gene.

We have constructed a chimeric class I gene in which the 5' half of the H-2Ld gene is linked to the 3' half of Q10d. The resulting H-2Ld/Q10d protein is homologous to the native H-2Ld heavy chain for the three external domains except for an Arg to His substitution at position 260. The transmembrane and intracytoplasmic domains of the H-2Ld chain are replaced by the short low hydrophobic transmembrane-like domain of the Q10d chain. Following DNA-mediated gene transfer into mouse L cells, transformants were selected for the presence of specific mRNA. Radiolabelling and immunoprecipitation analysis revealed secretion of a 48-46 kd chain weakly associated with beta 2-microglobulin. This molecule reacts with H-2Ld-specific mAb that identify determinants on the first and second domains as well as with an anti-Q10 carboxyl-terminal peptide antiserum, but is not recognized by a mAb specific for a determinant of H-2Ld third domain. The integrity of antibody reactivity of the first and second domains together with beta 2-microglobulin association suggest that our molecule may be considered a good soluble counterpart of the native membrane H-2Ld molecule with which to perform functional studies. In order to analyze the immunogenic capacities and T-cell recognition of the soluble H-2Ld molecules, T-cell lines were produced from mice of various inbred strains immunized with supernatant from H-2Ld/Q10d-transfected fibroblasts. Characterization of these T cells revealed that they expressed a CD4+CD8- phenotype, and recognized H-2Ld/Q10d products in a class II-restricted manner.

Animals↗

PubMed1999

Mytilus mitochondrial DNA contains a functional gene for a tRNASer(UCN) with a dihydrouridine arm-replacement loop and a pseudo-tRNASer(UCN) gene.

A 2500-nucleotide pair (ntp) sequence of F-type mitochondrial (mt) DNA of the Pacific Rim mussel Mytilus californianus (class Bivalvia, phylum Mollusca) that contains two complete (ND2 and ND3) and two partial (COI and COIII) protein genes and nine tRNA genes is presented. Seven of the encoded tRNAs (Ala, Arg, His, Met(AUA), Pro, Ser(UCN), and Trp) have the potential to fold into the orthodox four-armed tRNA secondary structure, while two [tRNASer(AGN) and a second tRNASer(UCN)] will fold only into tRNAs with a dihydrouridine (DHU) arm-replacement loop. Comparison of these mt-tRNA gene sequences with previously published, corresponding M. edulis F-type mtDNA indicates that similarity between the four-armed tRNASer(UCN) genes is only 63.8% compared with an average of 92.1% (range 86.2-98. 5%) for the remaining eight tRNA genes. Northern blot analysis indicated that mature tRNAs encoded by the DHU arm-replacement loop-containing tRNASer(UCN), tRNASer(AGN), tRNAMet(AUA), tRNATrp, and tRNAPro genes occur in M. californianus mitochondria, strengthening the view that all of these genes are functional. However, Northern blot and 5' RACE (rapid amplification of cDNA ends) analyses indicated that the four-armed tRNASer(UCN) gene is transcribed into a stable RNA that includes the downstream COI sequence and is not processed into a mature tRNA. On the basis of these observations the M. californianus and M. edulis four-armed tRNASer(UCN) sequences are interpreted as pseudo-tRNASer(UCN) genes.

Amino Acid Sequence↗

PubMed1986

Two dimensional polyacrylamide gel electrophoresis analysis of Tetrahymena mitochondrial tRNA.

Two dimensional (2D) urea-polyacrylamide gel electrophoresis of tRNA isolated from Tetrahymena mitochondria separated at least 36 spots, while more than 45 major and minor spots were resolved with cytosolic tRNA. Co-electrophoresis of mitochondrial and cytosolic tRNAs revealed that many spots co-migrate. When radioactive mitochondrial tRNA was hybridized to mtDNA under various conditions and tRNA melted from the hybrid was analyzed by 2D gel electrophoresis, only 10 tRNA spots were found. Identified as mtDNA-encoded were 2 spots for tRNA(leu), 2 for tRNA(met), and 1 each for tRNA(phe), tRNA(trp) and tRNA(tyr). The remaining three were unidentified. Mitochondrial tRNA spots that correspond to the tRNAs for arg, gly, ile, lys, ser, and val do not hybridize with mtDNA, and in gel positions they correspond to the cytoplasmic tRNA spots for the same respective amino acids. These mitochondrial tRNAs isolated from the gel can be acylated either by the mitochondrial or cytosolic enzymes. Mitochondrial tRNA isolated from a Tetrahymena cell homogenate which was pretreated with RNase A and Micrococcus nuclease exhibited the same 2D gel pattern as a non-treated control. Mitochondrial tRNAs from old and young cells showed generally similar tRNA spots in 2D gels, though more variable spots were seen with old cells. 3H-labeled whole-cell tRNA added to the cell homogenate prior to the mitochondrial isolation procedure did not remain associated with the final mitochondrial tRNA preparation. The present studies also showed mitochondrial tRNAs bound to the mitochondrial 80S monosome and polysome fractions. Radioactive tRNA added to the mitochondrial lysate does not adhere to the ribosomes, suggesting that the ribosome-bound tRNAs are not contaminating cytoplasmic tRNAs. These results are generally in good agreement with our previous data showing that only a small number of tRNAs are coded for by the mitochondrial DNA, while the others are a selected set of imported cytoplasmic tRNAs.

Amino Acids↗

PubMedDate not supplied

Activation of RNA polymerase I transcription by hepatitis C virus core protein.

The hepatitis C virus (HCV) core protein has been implicated in the transregulation of various RNA polymerase (Pol) II dependent genes as well as in the control of cellular growth and proliferation. In this study, we show that the core protein, whether individually expressed or produced as part of the HCV viral polyprotein, is the only viral product that has the potential to activate RNA Pol I transcription. Deletion analysis demonstrated that the fragment containing the N-terminal 1-156 residues, but not the 1-122 residues, of HCV core protein confers the same level of transactivation activity as the full-length protein. Moreover, the integrity of the Ser(116) and Arg(117) residues of HCV core protein was found to be critical for its transregulatory functions. We used DNA affinity chromatography to analyze the human ribosomal RNA promoter associated transcription machinery, and the results indicated that recruitment of the upstream binding factor and RNA Pol I to the ribosomal RNA promoter is enhanced in the presence of HCV core protein. Additionally, the HCV core protein mediated activation of ribosomal RNA transcription is accompanied by the hyperphosphorylation of upstream binding factor on serine residues, but not on threonine residues. Moreover, HCV core protein is present within the RNA Pol I multiprotein complex, indicating its direct involvement in facilitating the formation of a functional transcription complex. Protein-protein interaction studies further indicated that HCV core protein can associate with the selectivity factor (SL1) via direct contact with a specific component, TATA-binding protein (TBP). Additionally, the HCV core protein in cooperation with TBP is able to activate RNA Pol II and Pol III mediated transcription, in addition to RNA Pol I transcription. Thus, the results of this study suggest that HCV has evolved a mechanism to deregulate all three nuclear transcription systems, partly through targeting of the common transcription factor, TBP. Notably, the ability of the HCV core protein to upregulate RNA Pol I and Pol III transcription supports its active role in promoting cell growth, proliferation, and the progression of liver carcinogenesis during HCV infection.

Enzyme Activation↗

PubMed1993

Staphylococcus aureus has clustered tRNA genes.

The polymerase chain reaction (PCR) was used to detect large tRNA gene clusters in Bacillus subtilis, Bacillus badius, Bacillus megaterium, Lactobacillus brevis, Lactobacillus casei, and Staphylococcus aureus. The primers were based on conserved sequences of known gram-positive bacterial tRNA(Arg) and tRNA(Phe) genes. This PCR procedure detected an unusually large tRNA gene cluster in S. aureus. PCR-generated probes were used to identify a 4.5-kb EcoRI fragment that contained 27 tRNA genes immediately 3' to an rRNA operon. Some of these 27 tRNA genes are very similar, but only 1 is exactly repeated in the cluster. The 5' end of this cluster has a gene order similar to that found in the 9- and 21-tRNA gene clusters of B. subtilis. The 3' end of this S. aureus cluster exhibits more similarity to the 16-tRNA gene cluster of B. subtilis. The 24th, 25th, and 26th tRNA genes of this S. aureus tRNA gene cluster code for three similar, unusual Gly-tRNAs that may be used in the synthesis of the peptidoglycan in the cell wall but not in protein synthesis. Southern analysis of restriction digests of S. aureus DNA indicate that there are five to six rRNA operons in this bacterium's genome and that most or all may have large tRNA gene clusters at the 3' end.

Bacillus subtilis↗

PubMed2026

Differential contributions of mt-Tr and Cs variants to developmental cochlear defects and mitochondrial dysfunction in A/J mice.

A/J mice exhibit early-onset hearing loss linked to Cdh23, mitochondrial tRNA-Arg (mt-Tr), and citrate synthase (Cs) variants. Although developmental cochlear defects have been identified in juvenile A/J mice, the hierarchical contributions of mt-Tr versus Cs remain unclear. Using reciprocal intercross-derived strains to decouple mitochondrial haplotypes from nuclear factors, we demonstrate that the mitochondrial background is the primary determinant of auditory dysfunction. Mice with A/J mtDNA (AXB strains) displayed significantly higher ABR thresholds, accelerated hair cell attrition, and severe stereocilia dysmorphology compared to those with B6 mtDNA (BXA strains), occurring largely independently of the Cs genotype. While the Cs mutation exacerbated hearing loss, its impact was secondary to that of the dominant mitochondrial background. Systemic behavioral assessments and mitochondrial assays confirmed that A/J mitochondria exert a more profound metabolic impact than the Cs mutation. Our findings establish that the mitochondrial genomic background, with the mt-Tr locus as a prominent candidate variant, serves as the principal driver of developmental cochlear defects and early-onset hearing loss in A/J mice, while the nuclear Cs mutation acts as a synergistic modifier. This study underscores the critical role of mitonuclear crosstalk in inner ear maturation and provides new insights into the etiology of hereditary hearing loss.

Animals↗

PubMed1989

Variation in salmonid mitochondrial DNA: evolutionary constraints and mechanisms of substitution.

Sequence comparisons were made from 2214 bp of mitochondrial DNA cloned from six Pacific salmonid species. These sequences include the genes for ATPase subunit 6, cytochrome oxidase subunit 3, NADH dehydrogenase subunit 3, NADH dehydrogenase subunit 4L, tRNA(GLY), and tRNA(ARG). Variation is found at 338 silent and 12 nonsilent positions of protein coding genes and 10 positions in the two tRNA sequences. A single 3-bp length difference was also detected. In all pairwise comparisons the sequence divergence observed in the fragment was higher than that previously predicted by restriction enzyme analysis of the entire molecule. The inferred evolutionary relationship of these species is consistent between methods. The distribution of silent variation shows a complex pattern with greatly reduced variation at the junctions of genes. The variation in the tRNA sequences is concentrated in the DHU loop. The close relationship of these species and extensive sequence analyzed allows for an analysis of the spectrum of substitutions that includes the frequencies of all 12 possible substitutions. The observed spectrum of substitutions is related to potential pathways of spontaneous substitution. The salmonid sequences show an extremely high ratio of silent to replacement substitutions. In addition the amino acid sequences of the four proteins coded in this fragment show a consistently high level of identity with the Xenopus sequences. Taken together these data are consistent with a slower rate of amino acid substitution among the cold-blooded vertebrates when compared to mammals.

Adenosine Triphosphatases↗

PubMed1976

The involvement of the anticodon adjacent modified nucleoside N-(9-(BETA-D-ribofuranosyl) purine-6-ylcarbamoyl)-threonine in the biological function of E. coli tRNAile.

tRNAile was isolated from E. coli Cp 79 (leu-, arg-, thr-, his-, thiamin-, RCrel) which had been grown on a sub-optimal concentration of thr and was found to contain an average of 50% less N-[9-(beta-D-ribofuranosyl)- purin-6-ylcarbamoyl]threonine, t6Ado, than tRNAile from cells grown on an optimum concentration of thr and containing a normal complement of t6Ado. The two tRNA's were identical in their ability to be aminoacylated, to accept the 3'-terminal dinucleotide, and to form an ile-tRNAile-Tu-GTP complex. In contrast, the t6Ado-deficient-tRNA was significantly less efficient in binding to ribosomes compared to the normal tRNA. This difference was seen in the binding of deacylated tRNA and in the nonenzymatic and enzymatic binding of ile-tRNA, all in response to poly AUC. The t6Ado-deficient ile-tRNA demonstrated no binding at Mg2+ concentrations less than or equal to 10 mM, while the normal ile-tRNA bound at low Mg2+ concentrations. Tetracycline had the same effect on the normal as on the t6Ado-deficient ile-tRNA binding. As a control, the binding of phe-tRNA (which does not contain t6Ado) from normal and thr-starved cells in response to poly U was identical. It was concluded that t6Ado is required for proper codon-anticodon interaction.

Amino Acyl-tRNA Synthetases↗

PubMed2004

SsrA tagging of Escherichia coli SecM at its translation arrest sequence.

SecM is expressed from the secM-secA operon and activates the expression of secA in response to secretion defects. The 3'-end of secM encodes an "arrest sequence," which can interact with the ribosomal exit tunnel, preventing complete secM translation under secretion-defective conditions. In a cis-acting manner, ribosome stalling enhances secA translation. Pro(166) is the last residue incorporated when SecM elongation is arrested. We report that secretion deficiencies lead to SsrA tagging of SecM after Pro(166), Gly(165), and likely Arg(163). Northern blot analysis revealed the presence of a truncated secM transcript, likely issued from a secM-secA cleavage. The level of secM transcripts was decreased either when secM translation was totally prevented or when Pro(166) was mutated. However, the accumulation of a truncated secM transcript required secM translation and was prevented when the SecM arrest sequence was inactivated by a point mutation changing Pro(166) to Ala. We suggest that ribosome pausing at the site encoding the arrest sequence is required for formation of the truncated secM mRNA. SsrA tagging affected neither the presence of the secM mRNA nor secA expression, even under translocation-defective conditions. It is therefore likely that SsrA tagging of SecM occurs only after cleavage of secM-secA mRNA within the secM open reading frame encoding the SecM arrest sequence. Accumulation of transcripts expressing arrested SecM generated growth inhibition that was alleviated by the SsrA tagging system. Therefore, SsrA tagging of SecM would rescue ribosomes to avoid excessive jamming of the translation apparatus on stop-less secM mRNA.

Adenosine Triphosphatases↗

PubMed1999

A high throughput assay of the hepatitis C virus nonstructural protein 3 serine proteinase.

A simple assay was developed based on intramolecular fluorescence resonance energy transfer for detection of the activity of hepatitis C virus (HCV) serine proteinase. Two quenched-fluorogenic substrates, (7-methoxycoumarin-4-yl)acetyl (Mca) Asp-Asp-Ile-Val-Pro-Cys-Ser-Met-Ser-(2,4-dinitrophenyl, Dnp) Lys (Mca-Asp-Asp-Ile-Val-Pro-Cys-Ser-Met-Ser-Lys[Dnp], QF-1) and Mca-Asp-Asp-Ile-Val-Pro-Cys-Ser-Met-Lys(Dnp)-Arg-Arg (QF-2), which derived from the NS5A/5B junction of the HCV polyprotein, were designed. Kinetic studies revealed that QF-1 and QF-2 had high affinity for a recombinant enzyme which is a fusion protein of maltose binding protein and almost entire nonstructural protein (MBP-NS3), with Km values comparable to that of longer substrate based on the same cleavage site. QF-1 and QF-2 were cleaved by MBP-NS3 efficiently with kcat values of 7.5 and 4.2 min(-1), respectively. QF-2 was also found to be a good substrate of deltaNS3 which contained serine proteinase part of NS3 with kcat value of 4.3 min(-1). The cleavage reaction is detected continuously by the elevation of the fluorescence due to release from quenching. The fluorescence of the substrates increases in proportion to progress of the cleavage reaction under the standard conditions. This method was applied for screening of HCV serine protease inhibitors using a fluorescence multiwell plate reader. A group of natural occurring products, flavonoids, was subjected to be screened. Two flavonoids out of 25 were found to inhibit the enzyme moderately at a concentration of 100 microM. The data agreed with those obtained by high-performance liquid chromatography (HPLC). This method is suited to sensitive quantitation of the enzyme reaction as well as the high throughput analysis of the inhibitors.

Amino Acid Sequence↗

PubMed2002

Elevated systemic levels of endothelin-1 and blood pressure correlate with blunted constrictor responses and downregulation of endothelin(A), but not endothelin(B), receptors in an animal model of hypertension.

Hypertension is a major risk factor in the development of cardiovascular disease. Adenovirus gene transfer of endothelin-1 (Ad.CMV.ET-1) in rats produced significant (5-fold) increases in plasma ET-1 and systemic blood pressure (46%) 4 days after viral administration, compared with beta-galactosidase (Ad.CMV.beta-gal) injected as control. The density (B(max)) of the ET receptor ET(A) measured in aortas was reduced significantly by more than 50% to 17+/-2 fmol.mg(-1) of protein for the Ad.CMV.ET-1 group compared with 39+/-6 fmol x mg(-1) of protein for the control. There was no change in the density of the smaller population of the ET(B) sub-type. In agreement, the ratio of ET(A) mRNA to cyclophilin mRNA (a housekeeping gene) measured by Northern analysis was reduced in Ad.CMV.ET-1 rats compared with controls. The ratio of mRNA encoding the ET(B) sub-type did not change. ET-1 vasoconstriction was significantly reduced (P<0.05) in aortas from Ad.CMV.ET-1-treated rats [pD(2)=8.67+/-0.14 (where pD(2) is -log(10)EC(50)); n=11] versus the control (pD(2)=9.11+/-0.06; n=14) but there was no significant difference in the potency of two other vasoconstrictors tested (noradrenaline and Arg-vasopressin), indicating this was a specific effect on ET receptors. There was no change in the affinity of ET-1 binding to either receptor sub-type in the experimental group compared with the control, demonstrating that the attenuation in the constrictor response is the result of the reduced density of receptors rather than a change in affinity. The results show that ET(A) (but not ET(B)) receptors are modulated in this experimental model of hypertension and provide further evidence for selective blockade of the ET(A) receptor as a therapeutic strategy.

Animals↗

PubMed2004

Single amino acid changes in AspRS reveal alternative routes for expanding its tRNA repertoire in vivo.

Aminoacyl-tRNA synthetases (aaRSs) are enzymes that are highly specific for their tRNA substrates. Here, we describe the expansion of a class IIb aaRS-tRNA specificity by a genetic selection that involves the use of a modified tRNA displaying an amber anticodon and the argE(amber) and lacZ(amber) reporters. The study was performed on Escherichia coli aspartyl-tRNA synthetase (AspRS) and amber tRNA(Asp). Nine AspRS mutants able to charge the amber tRNA(Asp) and to suppress the reporter genes were selected from a randomly mutated library. All the mutants exhibited a new amber tRNA(Asp) specificity in addition to the initial native tRNA(Asp). Six mutations were found in the anticodon-binding site located in the N-terminal OB-fold. The strongest suppressor was a mutation of residue Glu-93 that contacts specifically the anticodon nucleotide 34 in the crystal structure. The other mutations in the OB-fold were found at close distance from the anticodon in the so-called loop L45 and strand S1. They concern residues that do not contact tRNA(Asp) in the native complex. In addition, this study shows that suppressors can carry mutations located far from the anticodon-binding site. One such mutation was found in the synthetase hinge-module where it increases the tRNA(Asp)-charging rate, and two other mutations were found in the prokaryotic-specific insertion domain and the catalytic core. These mutants seem to act by indirect effects on the tRNA acceptor stem binding and on the conformation of the active site of the enzyme. Altogether, these data suggest the existence of various ways for modifying the mechanism of tRNA discrimination.

Amino Acid Substitution↗