Differential import of nuclear-encoded tRNAGly isoacceptors into solanum Tuberosum mitochondria.
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In potato ( Solanum tuberosum ) mitochondria, about two-thirds of the tRNAs are encoded by the mitochondrial genome and one-third is imported from the cytosol. In the case of tRNAGly isoacceptors, a mitochondrial-encoded tRNAGly(GCC) was found in potato mitochondria, but this is likely to be insufficient to decode the four GGN glycine codons. In this work, we identified a cytosolic tRNAGly(UCC), which was found to be present in S.tuberosum mitochondria. The cytosolic tRNAGly(CCC) was also present in mitochondria, but to a lesser extent. By contrast, the cytosolic tRNAGly(GCC) could not be detected in mitochondria. This selective import of tRNAGly isoacceptors into S. tuberosum mitochondria raises further questions about the mechanism under-lying the specificity of the import process. (+info)
Mutations which alter the elbow region of tRNA2Gly reduce T4 gene 60 translational bypassing efficiency.
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Translating ribosomes bypass a 50 nucleotide coding gap in bacteriophage T4 gene 60 mRNA between codons 46 and 47 in order to synthesize the full-length protein. Bypassing of the coding gap requires peptidyl-tRNA2Gly detachment from a GGA codon (codon 46) followed by re-pairing at a matching GGA codon just before codon 47. Using negative selection, based on the sacB gene from Bacillus subtilis, Escherichia coli mutants were isolated which reduce bypassing efficiency. All of the mutations are in the gene for tRNA2Gly. Most of the mutations disrupt the hydrogen bonding interactions between the D- and T-loops (G18*psi55 and G19*C56) which stabilize the elbow region in nearly all tRNAs. The lone mutation not in the elbow region destabilizes the anticodon stem at position 40. Previously described Salmonella typhimurium mutants of tRNA2Gly, which reduce the stability of the T-loop, were also tested and found to decrease bypassing efficiency. Each tRNA2Gly mutant is functional in translation (tRNA2Gly is essential), but has a decoding efficiency 10- to 20-fold lower than wild-type. This suggests that rigidity of the elbow region and the anticodon stem is critical for both codon-anticodon stability and bypassing. (+info)
An extra tRNAGly(U*CU) found in ascidian mitochondria responsible for decoding non-universal codons AGA/AGG as glycine.
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Amino acid assignments of metazoan mitochondrial codons AGA/AGG are known to vary among animal species; arginine in Cnidaria, serine in invertebrates and stop in vertebrates. We recently found that in the mitochondria of the ascidian Halocynthia roretzi these codons are exceptionally used for glycine, and postulated that they are probably decoded by a tRNA(UCU). In order to verify this notion unambig-uously, we determined the complete RNA sequence of the mitochondrial tRNA(UCU) presumed to decode codons AGA/AGG in the ascidian mitochondria, and found it to have an unidentified U derivative at the anticodon first position. We then identified the amino acids attached to the tRNA(U*CU), as well as to the conventional tRNAGly(UCC) with an unmodified U34, in vivo. The results clearly demonstrated that glycine was attached to both tRNAs. Since no other tRNA capable of decoding codons AGA/AGG has been found in the mitochondrial genome, it is most probable that this tRNA(U*CU) does actually translate codons AGA/AGG as glycine in vivo. Sequencing of tRNASer(GCU), which is thought to recognize only codons AGU/AGC, revealed that it has an unmodified guanosine at position 34, as is the case with vertebrate mitochondrial tRNASer(GCU) for codons AGA/AGG. It was thus concluded that in the ascidian, codons AGU/AGC are read as serine by tRNASer(GCU), whereas AGA/AGG are read as glycine by an extra tRNAGly(U*CU). The possible origin of this unorthodox genetic code is discussed. (+info)
A novel tRNA-associated locus (trl) from Helicobacter pylori is co-transcribed with tRNA(Gly) and reveals genetic diversity.
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To date several genes have been identified in Helicobacter pylori that are expressed in only a proportion of strains, some of which are correlated with the pathogenicity of the bacterium. With this in mind, the present study was undertaken to identify other genes that are not expressed in all clinical isolates of H. pylori. Using arbitrarily primed PCR of RNA, a cDNA fragment of 187 bp (designated trl for transfer RNA-associated locus) was identified that was expressed in only one of two clinical isolates being tested. The fragment was purified, cloned and sequenced. A search of public databases prior to the release of the complete genome sequence of H. pylori strain 26695 showed no similarity with any other known genes or gene products. Inverse PCR was used to obtain further nucleotide sequence information surrounding the trl locus. A DNA probe derived from the trl locus hybridized with 32 (50%) of 64 clinical H. pylori isolates tested. Comparison of the nucleotide sequences of a trl-positive and trl-negative isolate showed that the locus is situated between two tRNA genes, tRNA(Gly) and tRNA(Leu), in H. pylori. Primer extension analysis showed that the trl locus is co-transcribed with tRNA(Gly). Analysis of the region between tRNA(Gly) and tRNA(Leu) in trl-negative isolates revealed additional genetic diversity among these isolates. (+info)
Complete DNA sequence of the mitochondrial genome of the ascidian Halocynthia roretzi (Chordata, Urochordata).
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The complete nucleotide sequence of the 14,771-bp-long mitochondrial (mt) DNA of a urochordate (Chordata)-the ascidian Halocynthia roretzi-was determined. All the Halocynthia mt-genes were found to be located on a single strand, which is rich in T and G rather than in A and C. Like nematode and Mytilus edulis mtDNAs, that of Halocynthia encodes no ATP synthetase subunit 8 gene. However, it does encode an additional tRNA gene for glycine (anticodon TCT) that enables Halocynthia mitochondria to use AGA and AGG codons for glycine. The mtDNA carries an unusual tRNA(Met) gene with a TAT anticodon instead of the usual tRNA(Met)(CAT) gene. As in other metazoan mtDNAs, there is not any long noncoding region. The gene order of Halocynthia mtDNA is completely different from that of vertebrate mtDNAs except for tRNA(His)-tRNA(Ser)(GCU), suggesting that evolutionary change in the mt-gene structure is much accelerated in the urochordate line compared with that in vertebrates. The amino acid sequences of Halocynthia mt-proteins deduced from their gene sequences are quite different from those in other metazoans, indicating that the substitution rate in Halocynthia mt-protein genes is also accelerated. (+info)
One protein from two open reading frames: mechanism of a 50 nt translational bypass.
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Translating ribosomes bypass a 50 nt coding gap in order to fuse the information found in the two open reading frames (ORFs) of bacteriophage T4 gene 60. This study investigates the underlying mechanism by focusing on the competition between initiation of bypassing and termination at the end of the first ORF. While nearly all ribosomes initiate bypassing, no more than 50% resume translation in the second ORF. Two previously described cis-acting stimulatory signals are critical for favoring initiation of bypassing over termination. Genetic analysis of these signals supports a working model in which the first (a stem-loop structure at the junction between the first ORF and the coding gap) interferes with decoding in the A-site, and the second (a stretch of amino acids in the nascent peptide encoded by the first ORF) destabilizes peptidyl-tRNA-mRNA pairing. (+info)
Sequence of the genome of the temperate, serotype-converting, Pseudomonas aeruginosa bacteriophage D3.
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Temperate bacteriophage D3, a member of the virus family Siphoviridae, is responsible for serotype conversion in its host, Pseudomonas aeruginosa. The complete sequence of the double-stranded DNA genome has been determined. The 56,426 bp contains 90 putative open reading frames (ORFs) and four genes specifying tRNAs. The latter are specific for methionine (AUG), glycine (GGA), asparagine (AAC), and threonine (ACA). The tRNAs may function in the translation of certain highly expressed proteins from this relatively AT-rich genome. D3 proteins which exhibited a high degree of sequence similarity to previously characterized phage proteins included the portal, major head, tail, and tail tape measure proteins, endolysin, integrase, helicase, and NinG. The layout of genes was reminiscent of lambdoid phages, with the exception of the placement of the endolysin gene, which parenthetically also lacked a cognate holin. The greatest sequence similarity was found in the morphogenesis genes to coliphages HK022 and HK97. Among the ORFs was discovered the gene encoding the fucosamine O-acetylase, which is in part responsible for the serotype conversion events. (+info)
Distinct modes of mature and precursor tRNA binding to Escherichia coli RNase P RNA revealed by NAIM analyses.
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We have analyzed by nucleotide analog interference mapping (NAIM) pools of precursor or mature tRNA molecules, carrying a low level of Rp-RMPalphaS (R = A, G, I) or Rp-c7-deaza-RMPalphaS (R = A, G) modifications, to identify functional groups that contribute to the specific interaction with and processing efficiency by Escherichia coli RNase P RNA. The majority of interferences were found in the acceptor stem, T arm, and D arm, including the strongest effects observed at positions G19, G53, A58, and G71. In some cases (interferences at G5, G18, and G71), the affected functional groups are candidates for direct contacts with RNase P RNA. Several modifications disrupt intramolecular tertiary contacts known to stabilize the authentic tRNA fold. Such indirect interference effects were informative as well, because they allowed us to compare the structural constraints required for ptRNA processing versus product binding. Our ptRNA processing and mature tRNA binding NAIM analyses revealed overlapping but nonidentical patterns of interference effects, suggesting that substrate binding and cleavage involves binding modes or conformational states distinct from the binding mode of mature tRNA, the product of the reaction. (+info)