Transfer RNA modification status influences retroviral ribosomal frameshifting.
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The possibility of whether tRNAs with and without a highly modified base in their anticodon loop may influence the level of retroviral ribosomal frameshifting was examined. Rabbit reticulocyte lysates were programmed with mRNA encoding UUU or AAC at the frameshift site and the corresponding Phe tRNA with or without the highly modified wyebutoxine (Y) base on the 3' side of its anticodon or Asn tRNA with or without the highly modified queuine (Q) base in the wobble position of its anticodon added. Phe and Asn tRNAs without the Y or Q base, respectively, stimulated the level of frameshifting, suggesting that the frameshift event is influenced by tRNA modification status. In addition, when AAU occurred immediately upstream of UUU as the penultimate frameshift site codon, addition of tRNAAsn without the Q base reduced the stimulatory effect of tRNAPhe without the Y base, whereas addition of tRNAAsn with the Q base did not alter the stimulatory effect. The addition of tRNAAsn without the Q base and tRNAPhe with the Y base inhibited frameshifting. The latter studies suggest an interplay between the tRNAs decoded at the penulimate frameshift and frameshift site codons that is also influenced by tRNA modification status. These data may be intrepreted as indicating that a hypomodified isoacceptor modulates frameshifting in an upward manner when utilized at the frameshift site codon, but modulates frameshifting in a downward manner when utilized at the penultimate frameshift site codon. (+info)
A cytotoxic ribonuclease targeting specific transfer RNA anticodons.
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The carboxyl-terminal domain of colicin E5 was shown to inhibit protein synthesis of Escherichia coli. Its target, as revealed through in vivo and in vitro experiments, was not ribosomes as in the case of E3, but the transfer RNAs (tRNAs) for Tyr, His, Asn, and Asp, which contain a modified base, queuine, at the wobble position of each anticodon. The E5 carboxyl-terminal domain hydrolyzed these tRNAs just on the 3' side of this nucleotide. Tight correlation was observed between the toxicity of E5 and the cleavage of intracellular tRNAs of this group, implying that these tRNAs are the primary targets of colicin E5. (+info)
Suppression of a mitochondrial tRNA gene mutation phenotype associated with changes in the nuclear background.
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We previously have characterized a pathogenic mtDNA mutation in the tRNAAsn gene. This mutation (G5703A) was associated with a severe mitochondrial protein synthesis defect and a reduction in steady-state levels of tRNAAsn. We now show that, although transmitochondrial cybrids harboring homoplasmic levels of the mutation do not survive in galactose medium, several galactose-resistant clones could be obtained. These cell lines had restored oxidative phosphorylation function and 2-fold higher steady-state levels of tRNAAsn when compared with the parental mutant cell line. The revertant lines contained apparently homoplasmic levels of the mutation and no other detectable alteration in the tRNAAsn gene. To investigate the origin of the suppression, we transferred mtDNA from the revertants (143B/206 TK-) to a different nuclear background (143B/207 TK-, 8AGr). These new transmitochondrial cybrids became defective once again in oxidative phosphorylation and regained galactose sensitivity. However, galactose-resistant clones could also be obtained by growing the 8AGr transmitochondrial cybrids under selection. Because the original rate of reversion was higher than that expected by a classic second site nuclear mutation, and because of the aneuploid features of these cell lines, we searched for the presence of chromosomal alterations that could be associated with the revertant phenotype. These studies, however, did not reveal any gross changes. Our results suggest that modulation of the dosage or expression of unknown nuclear-coded factor(s) can compensate for a pathogenic mitochondrial tRNA gene mutation, suggesting new strategies for therapeutic intervention. (+info)
Yeast asparagine (Asn) tRNA without Q base promotes eukaryotic frameshifting more efficiently than mammalian Asn tRNAs with or without Q base.
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In this study, we compare the efficiency of Asn tRNA from mammalian sources with and without the highly modified queuosine (Q) base in the wobble position of its anticodon and Asn tRNA from yeast, which naturally lacks Q base, to promote frameshifting. Interestingly, no differences in the ability of the two mammalian Asn tRNAs to promote frameshifting were observed, while yeast tRNA(ASn)(-Q) promoted frameshifting more efficiently than its mammalian counterparts in both rabbit reticulocyte lysates and wheat germ extracts. The shiftability of yeast Asn tRNA is therefore not due, or at least not completely, to the lack of Q base and most likely the shiftiness resides in structural differences elsewhere in the molecule. However, we cannot absolutely rule out a role of Q base in frameshifting as wheat germ extracts and a lysate depleted of most of its tRNA and supplemented with calf liver tRNA contain both Asn tRNA with or without Q base. (+info)
A single amidotransferase forms asparaginyl-tRNA and glutaminyl-tRNA in Chlamydia trachomatis.
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Aminoacyl-tRNA is generally formed by aminoacyl-tRNA synthetases, a family of 20 enzymes essential for accurate protein synthesis. However, most bacteria generate one of the two amide aminoacyl-tRNAs, Asn-tRNA or Gln-tRNA, by transamidation of mischarged Asp-tRNA(Asn) or Glu-tRNA(Gln) catalyzed by a heterotrimeric amidotransferase (encoded by the gatA, gatB, and gatC genes). The Chlamydia trachomatis genome sequence reveals genes for 18 synthetases, whereas those for asparaginyl-tRNA synthetase and glutaminyl-tRNA synthetase are absent. Yet the genome harbors three gat genes in an operon-like arrangement (gatCAB). We reasoned that Chlamydia uses the gatCAB-encoded amidotransferase to generate both Asn-tRNA and Gln-tRNA. C. trachomatis aspartyl-tRNA synthetase and glutamyl-tRNA synthetase were shown to be non-discriminating synthetases that form the misacylated tRNA(Asn) and tRNA(Gln) species. A preparation of pure heterotrimeric recombinant C. trachomatis amidotransferase converted Asp-tRNA(Asn) and Glu-tRNA(Gln) into Asn-tRNA and Gln-tRNA, respectively. The enzyme used glutamine, asparagine, or ammonia as amide donors in the presence of either ATP or GTP. These results suggest that C. trachomatis employs the dual specificity gatCAB-encoded amidotransferase and 18 aminoacyl-tRNA synthetases to create the complete set of 20 aminoacyl-tRNAs. (+info)
RSEARCH: finding homologs of single structured RNA sequences.
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BACKGROUND: For many RNA molecules, secondary structure rather than primary sequence is the evolutionarily conserved feature. No programs have yet been published that allow searching a sequence database for homologs of a single RNA molecule on the basis of secondary structure. RESULTS: We have developed a program, RSEARCH, that takes a single RNA sequence with its secondary structure and utilizes a local alignment algorithm to search a database for homologous RNAs. For this purpose, we have developed a series of base pair and single nucleotide substitution matrices for RNA sequences called RIBOSUM matrices. RSEARCH reports the statistical confidence for each hit as well as the structural alignment of the hit. We show several examples in which RSEARCH outperforms the primary sequence search programs BLAST and SSEARCH. The primary drawback of the program is that it is slow. The C code for RSEARCH is freely available from our lab's website. CONCLUSION: RSEARCH outperforms primary sequence programs in finding homologs of structured RNA sequences. (+info)
Using a solid-phase ribozyme aminoacylation system to reprogram the genetic code.
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Here, we report a simple and economical tRNA aminoacylation system based upon a resin-immobilized ribozyme, referred to as Flexiresin. This catalytic system features a broad spectrum of activities toward various phenylalanine (Phe) analogs and suppressor tRNAs. Most importantly, it allows users to perform the tRNA aminoacylation reaction and isolate the aminoacylated tRNAs in a few hours. We coupled the Flexiresin system with a high-performance cell-free translation system and demonstrated protein mutagenesis with seven different Phe analogs in parallel. Thus, the technology developed herein provides a new tool that significantly simplifies the procedures for the synthesis of aminoacyl-tRNAs charged with nonnatural amino acids, which makes the nonnatural amino acid mutagenesis of proteins more user accessible. (+info)
Organization of the mitochondrial genomes of whiteflies, aphids, and psyllids (Hemiptera, Sternorrhyncha).
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BACKGROUND: With some exceptions, mitochondria within the class Insecta have the same gene content, and generally, a similar gene order allowing the proposal of an ancestral gene order. The principal exceptions are several orders within the Hemipteroid assemblage including the order Thysanoptera, a sister group of the order Hemiptera. Within the Hemiptera, there are available a number of completely sequenced mitochondrial genomes that have a gene order similar to that of the proposed ancestor. None, however, are available from the suborder Sternorryncha that includes whiteflies, psyllids and aphids. RESULTS: We have determined the complete nucleotide sequence of the mitochondrial genomes of six species of whiteflies, one psyllid and one aphid. Two species of whiteflies, one psyllid and one aphid have mitochondrial genomes with a gene order very similar to that of the proposed insect ancestor. The remaining four species of whiteflies had variations in the gene order. In all cases, there was the excision of a DNA fragment encoding for cytochrome oxidase subunit III(COIII)-tRNAgly-NADH dehydrogenase subunit 3(ND3)-tRNAala-tRNAarg-tRNAasn from the ancestral position between genes for ATP synthase subunit 6 and NADH dehydrogenase subunit 5. Based on the position in which all or part of this fragment was inserted, the mitochondria could be subdivided into four different gene arrangement types. PCR amplification spanning from COIII to genes outside the inserted region and sequence determination of the resulting fragments, indicated that different whitefly species could be placed into one of these arrangement types. A phylogenetic analysis of 19 whitefly species based on genes for mitochondrial cytochrome b, NADH dehydrogenase subunit 1, and 16S ribosomal DNA as well as cospeciating endosymbiont 16S and 23S ribosomal DNA indicated a clustering of species that corresponded to the gene arrangement types. CONCLUSIONS: In whiteflies, the region of the mitochondrial genome consisting of genes encoding for COIII-tRNAgly-ND3-tRNAala-tRNAarg-tRNAasn can be transposed from its ancestral position to four different locations on the mitochondrial genome. Related species within clusters established by phylogenetic analysis of host and endosymbiont genes have the same mitochondrial gene arrangement indicating a transposition in the ancestor of these clusters. (+info)