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At least 145 records · Page 8Linked to original sources
PubMed1999

Selection of cleavage site by mammalian tRNA 3' processing endoribonuclease.

Mammalian tRNA 3' processing endoribonuclease (3' tRNase) removes 3' trailers from pre-tRNAs by cleaving the RNA immediately downstream of the discriminator nucleotide. Although 3' tRNase can recognize and cleave any target RNA that forms a pre-tRNA-like complex with another RNA, in some cases cleavage occurs at multiple sites near the discriminator. We investigated what features of pre-tRNA determine the cleavage site using various pre-tRNAArg variants and purified pig enzyme. Because the T stem-loop and the acceptor stem plus a 3' trailer are sufficient for recognition by 3' tRNase, we constructed variants that had additions and/or deletions of base-pairs in the T stem and/or the acceptor stem. Pre-tRNAs lacking one and two acceptor stem base-pairs were cleaved one and two nucleotides and two and three nucleotides, respectively, downstream of the discriminator. On the other hand, pre-tRNA variants containing extra acceptor stem base-pairs were cleaved only after the discriminator. The cleavage site was shifted to one and two nucleotides downstream of the discriminator by deleting one base-pair from the T stem, but was not changed by additional base-pairs in the T stem. Pre-tRNA variants that contained an eight base-pair acceptor stem plus a six base-pair T stem, an eight base-pair acceptor stem plus a four base-pair T stem, or a six base-pair acceptor stem plus a six base-pair T stem were all cleaved after the original nucleotide. In general, pre-tRNA variants containing a total of more than 11 bp in the acceptor stem and the T stem were cleaved only after the discriminator, and pre-tRNA variants with a total of N bp (N is less than 12) were cleaved 12-N and 13-N nt downstream of the discriminator. Cleavage efficiency of the variants decreased depending on the degree of structural changes from the authentic pre-tRNA. This suggests that the numbers of base-pairs of both the acceptor stem and the T stem are important for recognition and cleavage by 3' tRNase.

Animals↗
PubMed1999

Expression and characterization of the HMG-CoA reductase of the thermophilic archaeon Sulfolobus solfataricus.

The thermostable class I HMG-CoA reductase of Sulfolobus solfataricus offers potential for industrial applications and for the initiation of crystallization trials of a biosynthetic HMG-CoA reductase. However, of the 15 arginine codons of the hmgA gene that encodes S. solfataricus HMG-CoA reductase, 14 (93%) are AGA or AGG, the arginine codons used least frequently by Escherichia coli. The presence of these rare codons in tandem or in the first 20 codons of a gene can complicate expression of that gene in E. coli. Problems include premature chain termination and misincorporation of lysine for arginine. We therefore sought to improve the expression and subsequent yield of S. solfataricus HMG-CoA reductase by expanding the pool size of tRNA(AGA,AGG), the tRNA that recognizes these two rare codons. Coexpression of the S. solfataricus hmgA gene with the argU gene that encodes tRNA(AGA,AGG) resulted in an over 10-fold increase in enzyme yield. This has provided significantly greater quantities of purified enzyme for potential industrial applications and for crystallographic characterization of a stable class I HMG-CoA reductase. It has, in addition, facilitated determination of kinetic parameters and of pH optima for all four catalyzed reactions, for determination of the K(i) for inhibition by the statin drug mevinolin, and for comparison of the properties of the HMG-CoA reductase of this thermophilic archaeon to those of other class I HMG-CoA reductases.

Acyl Coenzyme A↗
PubMed1990

Identification and characterization of a defective SSV1 genome integrated into a tRNA gene in the archaebacterium Sulfolobus sp. B12.

Within the chromosome of the archaebacterium Sulfolobus sp. B12, a 7.4 kb region was identified which displayed extensive sequence similarities to the 15.5 kb genetic element SSV1 carried by the same strain both as a circular form and as a site-specifically integrated copy. DNA sequence analysis indicated that this 7.4 kb region (designated SSV1intB) represented an SSV1-like element distinguishable from the full-length integrated copy (designated SSV1intA) by extensive deletions and point mutations. The physical organization of DNA sequences of SSV1intB indicated that this element was integrated at the same attP site as previously identified for SSV1intA. A comparison of the DNA sequences at the left attachment sites of SSV1intA and SSV1intB revealed that they both represented very similar putative arginine tRNA genes followed by a 10 bp inverted repeat sequence. S1 nuclease mapping experiments indicated that these tRNA genes are transcribed.

Archaea↗
PubMed1994

A ribosomal frameshifting error during translation of the argI mRNA of Escherichia coli.

Using fusions between the Escherichia coli genes argI and lacZ, it has been demonstrated that ribosomal frameshifting occurs at a frequency of between 3% and 16% within the argI mRNA, soon after the initiation codon. The frameshift involves a phenylalanyl-tRNA shifting into the +1 frame at the sequence UUU-U/C. The shift does not occur if the in-frame phenylalanine codon UUU is replaced by UUC. The level of frameshifting is higher in dense cultures and is not dependent on phenylalanine starvation. In the wild-type argI gene this frameshifting event would be an error, leading to a truncated, non-functional protein. Therefore, it is unlike the numerous examples of required frameshifting events that have been described in other genes.

Amino Acid Sequence↗
PubMed1994

Serine protease inhibitors block N-terminal arginylation of proteins by inhibiting the arginylation of tRNA in rat brains.

The tRNA mediated, posttranslational, N-terminal arginylation of proteins occurs in all eukaryotic cells. In nervous tissue, these reactions can be inhibited by endogenous molecules with a molecular weight of between one thousand and five thousand. In the present experiments, exogenous serine protease inhibitors (10(-5) M or less) but not other types of protease inhibitors, were found to be able to block the arginylation of protein in extracts of rat brain homogenates. Inhibition was not by the usual mode of action of protease inhibitors, but by interfering (non-competitively) with the charging of tRNA. Since arginylated proteins are rapidly ubiquitinated and degraded by cytosolic proteases, serine protease inhibitors may act to stabilize proteins by a dual mechanism of inhibiting arginylation as well as inhibiting serine proteases.

Animals↗
PubMed1988

Sequence and transcriptional analysis of a chloroplast insert in the mitochondrial genome of Zea mays.

The complete sequence of a mitochondrial DNA insertional event containing the 3' portion of the chloroplast 23S-4.5S rRNA gene, the entire 5S rRNA gene and intervening sequence and all but the 3' 6 nucleotides of the arginine tRNA gene is reported. Also reported are both chloroplast/mitochondrial DNA junction sequences, 551 nucleotides of flanking mitochondrial sequences and the genomic location of this insert in Zea mays mitochondria. Utilizing the distinctive transcriptional pattern seen for mitochondrial RNA derived from root tissue relative to shoot tissue, we also reported a general experimental test for whether chloroplast sequences transposed to the mitochondrion are transcribed. Although results for the insert reported suggest it is transcriptionally inactive, the technique should be generally applicable to any transposed sequence.

Base Sequence↗
PubMed1998

High-level expression of murine terminal deoxynucleotidyl transferase in Escherichia coli grown at low temperature and overexpressing argU tRNA.

Terminal deoxynucleotidyl transferase (TdT) is a highly conserved vertebrate enzyme that possesses the unique ability to catalyze the random addition of deoxynucleoside 5'-triphosphates onto the 3'-hydroxyl group of a single-stranded DNA. It plays an important role in the generation of immunoglobin and T-cell receptor diversity. TdT is usually obtained from animal thymus gland or produced in a baculovirus system, but both procedures are rather tedious, and proteolysis occurs during purification. Attempts to overexpress TdT in bacteria have been unsuccessful or have yielded an enzyme with a lower specific activity. A dearth of TdT has thus hampered detailed structural and functional studies. In the present study, we report that by lowering growth temperature and overexpressing a rare arginyl tRNA, it is possible to boost the production in Escherichia coli of murine TdT with minimal proteolysis and high specific activity.

Animals↗
PubMed1998

Transcription of plastid-derived tRNA genes in rice mitochondria.

Previous investigations located nine of the genes for rice tRNAs on plastid (pt)-derived sequences in mitochondrial DNA. In the present study, we examined whether these genes were also transcribed in rice mitochondria. Northern-blot hybridization revealed that seven of these genes (trnC, trnF, trnH, trnM, trnN, trnS and trnW) are transcribed and are precisely processed to mature tRNAs. One of the other two genes (trnP) is transcribed but cannot be processed efficiently, while the other (trnR), which has 100% identity to the original plastid tRNAArg gene, is not transcribed in rice mitochondria. These results suggest that seven of the nine pt-derived tRNAs may be utilized for the biosynthesis of protein in plant mitochondria.

DNA Probes↗
PubMed1997

The Brevibacterium albidum gene encoding the arginine tRNACCG complements the growth defect of an Escherichia coli strain carrying a thermosensitive mutation in the rnpA gene at the nonpermissive temperature.

The Escherichia coli rnpA gene encodes C5 protein, the protein component of RNase P. The rnpA49 mutation renders the C5 protein thermosensitive, which results in thermosensitivity of RNase P function. The chromosomal DNA region from Brevibacterium albidum that complements the rnpA49 mutation was analysed. The gene capable of complementing the growth defect of an rnpA49 mutant strain at nonpermissive temperature was identified as the gene for an arginine tRNA with anticodon CCG by a deletion analysis combined with complementation assays. Transcription of the arginine tRNA gene carried on a multicopy plasmid was correlated with the complementation of the rnpA49 mutation, indicating that the gene product is indeed responsible for complementation of the rnpA49 mutation.

Base Sequence↗
PubMed1992

Importance of mRNA folding and start codon accessibility in the expression of genes in a ribosomal protein operon of Escherichia coli.

The trmD operon of Escherichia coli consists of the genes for the ribosomal protein (r-protein) S16, a 21 kilodalton protein (21K) of unknown function, the tRNA(m1G37)methyltransferase (TrmD), and r-protein L19, in that order. The synthesis of the 21K and TrmD proteins is 12 and 40-fold lower, respectively, than that of the two r-proteins, although the corresponding parts of the mRNA are equally abundant. This translational control of expression of at least the 21K protein gene (21K), is mediated by a negative control element located between codons 18 and 50 of 21K. Here, we present evidence for a model in which mRNA sequences up to around 100 nucleotides downstream from the start codon of 21K fold back and base-pair to the 21K translation initiation region, thereby decreasing the translation initiation frequency. Mutations in the internal negative control element of 21K that would prevent the formation of the proposed mRNA secondary structure over both the Shine-Dalgarno (SD) sequence and the start codon increased expression up to about 20-fold, whereas mutations that would disrupt the base-pairing with the SD-sequence had only relatively small effects on expression. In addition, the expression increased 12-fold when the stop codon of the preceding gene, rpsP, was moved next to the SD-sequence of 21K allowing the ribosomes to unfold the postulated mRNA secondary structure. The expression increased up to 150-fold when that stop codon change was combined with the internal negative control element base-substitutions that derepressed translation about 20-fold. The negative control element of 21K does not seem to be responsible for the low expression of the trmD gene located downstream. However, a similar negative control element native to trmD can explain at least partly the low expression of trmD. Possibly, the two mRNA secondary structures function to decouple translation of 21K and trmD from that of the respective upstream cistron in order to achieve their independent regulation.

Base Sequence↗
PubMed2026

Dietary arginine drives codon-dependent MHC class I translation and improves immunity in colon tumorigenesis and respiratory viral infection.

Amino acid levels fluctuate across diverse pathological conditions. Whether such amino acid modulations directly shape pathophysiology by regulating host gene expression remains unknown. We found that extracellular arginine restriction, observed in cancer and infection, represses specific arginine tRNAs-directly suppressing translation of major histocompatibility complex I (MHC class I) and antigen presentation. Arginine regulation of MHC class I was codon-usage dependent, as synonymous codon mutations prevented MHC class I modulation. Dietary arginine restriction impaired anti-viral immunity against influenza and SARS-CoV-2 and increased colon tumorigenesis. Conversely, increasing arginine availability via dietary supplementation or myeloid-specific arginase 1 deletion enhanced MHC class I protein levels, suppressed colon tumorigenesis, and improved viral infection outcomes. These disease-modulating effects were abolished in β2-microglobulin (B2m)-deficient mice. Thus, dietary modulation of a single amino acid critically influences codon-biased translation and MHC class I-mediated immunity to respiratory viral infections and cancer, revealing an unexpected mechanism and disease hazard for arginine deficiency and highlighting potential for amino acid-based translation modulation therapy.

Animals↗
PubMed2006

Identification and characterization of a +1 frameshift observed during the expression of Epstein-Barr virus IL-10 in Escherichia coli.

Epstein-Barr virus IL-10 (ebvIL-10) mimics the biological functions of cellular IL-10 including a number of immunoinhibitory activities on diverse immune cells. Characterization of ebvIL-10 and several mutants, expressed in Escherichia coli, by gel filtration chromatography and mass spectrometry revealed a +1 frameshift upon ebvIL-10 expression. The frameshift is caused by the rare AGG codon at ebvIL-10 Arg159, which is followed by the most inefficient stop signal, UGAC. The frameshift was corrected by substituting the rare AGG codon with an abundant arginine codon, CGU, or by enhancing the level of tRNA that decodes the AGG codon. As a result, ebvIL-10 expression levels increased by approximately 3-fold and the purity of the protein improved from 85-95% to 98-99%. The correction of the frameshift has been essential for continuing structural and biophysical studies of ebvIL-10.

Amino Acid Sequence↗
PubMed2006

Molecular systematics of Vampyressine bats (Phyllostomidae: Stenodermatinae) with comparison of direct and indirect surveys of mitochondrial DNA variation.

Approximately 29 species in seven genera (Chiroderma, Mesophylla, Platyrrhinus, Uroderma, Vampyressa, Vampyriscus, and Vampyrodes) compose the Subtribe Vampyressina, a group of New World leaf-nosed bats (Phyllostomidae) specialized in fruit-eating. A recent study of restriction-site variability within the mitochondrial ND3-ND4 gene region contrasts with other molecular data, including sequence data from other mitochondrial genes, by suggesting that the monotypic genus Ectophylla (E. alba) also is member of the group and is related closely to Mesophylla. In this study, we address possible explanations for why the restriction-site data appear to contradict other molecular data by performing phylogenetic analysis of DNA sequence variation (direct survey) in the ND3-ND4 region and cytochrome b gene and by re-assessing ND3-ND4 restriction-site variability in the known sequences (indirect survey). Results from analysis of sequence data reject the Ectophylla-Mesophylla hypothesis (P<0.001) and suggest four primary lineages within Vampyressina: (1) Mesophylla-Vampyressa; (2) Chiroderma-Vampyriscus; (3) Platyrrhinus-Vampyrodes; and (4) Uroderma. We also find no support for the Ectophylla-Mesophylla hypothesis in our re-analysis of ND3-ND4 restriction-site variability, and suggest the differences between molecular studies have a methodological basis.

Animals↗
PubMed1999

High level expression of Thermococcus litoralis 4-alpha-glucanotransferase in a soluble form in Escherichia coli with a novel expression system involving minor arginine tRNAs and GroELS.

The Thermococcus litoralis 4-alpha-glucanotransferase (GTase) gene has a high content of AGA and AGG codons for arginine, which are extremely rare in Escherichia coli. Expression of the GTase gene in E. coli resulted in low protein production and the accumulation of inclusion bodies. However, simultaneous expression of GTase with tRNA(AGA), tRNA(AGG) and GroELS affected both the production and solubility of GTase, and production of soluble GTase increasing about 5-fold. This new E. coli expression system should be applicable to the expression of not only archaeal but also eukaryotic genes, which usually contain a large number of AGA and AGG codons.

Chaperonin 60↗
PubMed1991

Upstream sequence activation of Escherichia coli argT promoter in vivo and in vitro.

Escherichia coli argT promoter in a galK fusion construct is shown by BAL 31 deletion to require its upstream region for high in vivo activity. The extent of activation conferred by the upstream sequence from -130 to -38 is 25-fold. A spontaneous mutant containing a T to G transversion at -37 (i.e., the T-37G promoter) shows a similar requirement; however, the upstream sequence producing a 10-fold effect spans only -130 to -60. The difference in upstream sequence boundaries between the wild-type and T-37G promoters suggests the possible existence of two activating elements. Gel mobility investigation points to the presence of bent DNA in the argT promoter, and the bent center was localized to the -90 to -95 region by circular permutation analysis. The role of the upstream activating sequence (UAS) in promoter function was probed by competitive transcription experiments in vitro. Results of this type of analysis indicate that the full UAS activates transcription through a combined effect on KB and k2. Of these, KB is significantly strengthened by the proximal element, and k2 is stimulated to a smaller extent by the distal element. The evidence from deletion analysis, gel mobility investigation, and competitive transcription together support a "two-element" model of UAS function for the argT promoter.

Base Sequence↗
PubMed2000

Biochemical and molecular basis for mitochondrial cardiomyopathy in neonates and children.

Defects in myocardial bioenergetics have been reported in patients with cardiomyopathy but their molecular basis and role in pathophysiology remain unclear. We sought to establish a molecular basis for cardiac mitochondrial respiratory enzyme abnormalities frequently present (75%) in a group of 16 children (including 2 neonates) with end-stage cardiomyopathy. Decreased specific activity levels were found in complexes I, III, IV and V but not in II, the only complex that is entirely nuclear encoded. Sequence analysis of cardiac mtDNA revealed 4 patients harbouring heteroplasmic mtDNA mutations in cytb, tRNAArg, and ND5 at highly conserved positions. These mutations were present neither in controls nor in patients without enzymatic defect. In addition, 4 patients exhibited marked reduction in cardiac mtDNA levels. The basis for respiratory enzyme abnormalities can be explained in a subset of our patients as a result of either pathogenic mtDNA mutation or depletion. Patients harbouring both DNA and enzymatic defects fulfil rigorous criteria defining mitochondrial cardiomyopathy.

Adenosine Triphosphatases↗
PubMed2004

Structure of a purine-purine wobble base pair in the decoding center of the ribosome.

Here we report the crystal structures of I.C and I.A wobble base pairs in the context of the ribosomal decoding center, clearly showing that the I.A base pair is of an I(anti).A(anti) conformation, as predicted by Crick. Additionally, the structures enable the observation of changes in the anticodon to allow purine-purine base pairing, the 'widest' base pair geometry allowed in the wobble position.

Anticodon↗