Protein-protein, protein-RNA and protein-lipid interactions of signal-recognition particle components in the hyperthermoacidophilic archaeon Acidianus ambivalens.
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The signal-recognition particle (SRP) of one of the most acidophilic and hyperthermophilic archaeal cells, Acidianus ambivalens, and its putative receptor component, FtsY (prokaryotic SRP receptor), were investigated in detail. A. ambivalens Ffh (fifty-four-homologous protein) was shown to be a soluble protein with strong affinity to membranes. In its membrane-residing form, Ffh was extracted from plasma membranes with chaotropic agents like urea, but not with agents diminishing electrostatic interactions. Using unilamellar tetraether phospholipid vesicles, both Ffh and FtsY associate independently from each other in the absence of other factors, suggesting an equilibrium of soluble and membrane-bound protein forms under in vivo conditions. The Ffh protein precipitated from cytosolic cell supernatants with anti-Ffh antibodies, together with an 7 S-alike SRP-RNA, suggesting a stable core ribonucleoprotein composed of both components under native conditions. The SRP RNA of A. ambivalens depicted a size of about 309 nucleotides like the SRP RNA of the related organism Sulfolobus acidocaldarius. A stable heterodimeric complex composed of Ffh and FtsY was absent in cytosolic supernatants, indicating a transiently formed complex during archaeal SRP targeting. The FtsY protein precipitated in cytosolic supernatants with anti-FtsY antisera as a homomeric protein lacking accessory protein components. However, under in vitro conditions, recombinantly generated Ffh and FtsY associate in a nucleotide-independent manner, supporting a structural receptor model with two interacting apoproteins. (+info)
Analysis of the open region and of DNA-protein contacts of archaeal RNA polymerase transcription complexes during transition from initiation to elongation.
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The archaeal transcriptional machinery is polymerase II (pol II)-like but does not require ATP or TFIIH for open complex formation. We have used enzymatic and chemical probes to follow the movement of Pyrococcus RNA polymerase (RNAP) along the glutamate dehydrogenase gene during transcription initiation and transition to elongation. RNAP was stalled between registers +5 and +20 using C-minus cassettes. The upstream edge of RNAP was in close contact with the archaeal transcription factors TATA box-binding protein/transcription factor B in complexes stalled at position +5. Movement of the downstream edge of the RNAP was not detected by exonuclease III footprinting until register +8. A first structural transition characterized by movement of the upstream edge of RNAP was observed at registers +6/+7. A major transition was observed at registers +10/+11. In complexes stalled at these positions also the downstream edge of RNA polymerase started translocation, and reclosure of the initially open complex occurred indicating promoter clearance. Between registers +11 and +20 both RNAP and transcription bubble moved synchronously with RNA synthesis. The distance of the catalytic center to the front edge of the exo III footprint was approximately 12 nucleotides in all registers. The size of the RNA-DNA hybrid in an early archaeal elongation complex was estimated between 9 and 12 nucleotides. For complexes stalled between positions +10 and +20 the size of the transcription bubble was around 17 nucleotides. This study shows characteristic mechanistic properties of the archaeal system and also similarities to prokaryotic RNAP and pol II. (+info)
Recognition of tRNA by aminoacyl-tRNA synthetase from hyperthermophilic archaea, Aeropyrum pernix K1.
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To study the recognition sites of tRNA for archaeal aminoacyl-tRNA synthetase, several aminoacyl-tRNA synthetase genes from hyperthermophilic archaeon, Aeropyrum pernix K1 were cloned and expressed. All the expressed enzymes showed extreme thermostability. Expressed threonyl-tRNA synthetase threonylated not only archaeal (A. pernix and Haloferax volcanii) threonine tRNAs but also Escherichia coli threonine tRNA. However, threonyl-tRNA synthetase from H. volcanii did not threonylate E. coli threonine tRNA. (+info)
Efficient RNA 2'-O-methylation requires juxtaposed and symmetrically assembled archaeal box C/D and C'/D' RNPs.
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Box C/D ribonucleoprotein (RNP) complexes direct the nucleotide-specific 2'-O-methylation of ribonucleotide sugars in target RNAs. In vitro assembly of an archaeal box C/D sRNP using recombinant core proteins L7, Nop56/58 and fibrillarin has yielded an RNA:protein enzyme that guides methylation from both the terminal box C/D core and internal C'/D' RNP complexes. Reconstitution of sRNP complexes containing only box C/D or C'/D' motifs has demonstrated that the terminal box C/D RNP is the minimal methylation-competent particle. However, efficient ribonucleotide 2'-O-methylation requires that both the box C/D and C'/D' RNPs function within the full-length sRNA molecule. In contrast to the eukaryotic snoRNP complex, where the core proteins are distributed asymmetrically on the box C/D and C'/D' motifs, all three archaeal core proteins bind both motifs symmetrically. This difference in core protein distribution is a result of altered RNA-binding capabilities of the archaeal and eukaryotic core protein homologs. Thus, evolution of the box C/D nucleotide modification complex has resulted in structurally distinct archaeal and eukaryotic RNP particles. (+info)
Caldisphaera lagunensis gen. nov., sp. nov., a novel thermoacidophilic crenarchaeote isolated from a hot spring at Mt Maquiling, Philippines.
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Four novel, thermoacidophilic, crenarchaeotic cocci that grew anaerobically and heterotrophically were isolated from an acidic hot spring in the Philippines; two representative strains were characterized in detail. Most cells were regular cocci, 0.8-1.1 microm in width, which occurred singly or in pairs. They were non-motile and grew at 45-80 degrees C (optimum 70-75 degrees C) and pH 2.3-5.4 (optimum 3.5-4.0). They utilized starch, glycogen, gelatin, beef extract, yeast extract and peptone as carbon and energy sources. Growth was stimulated by the presence of sulfur as an electron acceptor. The lipid fraction contained cyclic and acyclic tetraether core lipids. The DNA G + C content was 31 mol%; phylogenetic analysis based on 16S rDNA sequences showed that the novel cocci represent an independent lineage in the phylum Crenarchaeota, distantly related to Acidilobus aceticus and an allied strain, NC12. Caldisphaera lagunensis gen. nov., sp. nov. is proposed to accommodate the four strains. The type strain is IC-154T (=JCM 11604T=MCC-UPLB 1331T=ANMR 0165T). (+info)
Threonyl-tRNA synthetase of archaea: importance of the discriminator base in the aminoacylation of threonine tRNA.
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To investigate the contribution of the discriminator base of archaeal tRNA(Thr) in aminoacylation by threonyl-tRNA synthetase (ThrRS), cross-species aminoacylation between Escherichia coli and Haloferax volcanii, halophilic archaea, was studied. It was found that E. coli ThrRS threonylated the H. volcanii tRNA(Thr) but that E. coli threonine tRNA was not aminoacylated by H. volcanii ThrRS. Results of a threonylation experiment using in vitro mutants of E. coli threonine tRNA showed that only the mutant tRNA(Thr) having U73 was threonylated by H. volcanii ThrRS. These findings indicate that the discriminator base U73 of H. volcanii tRNA(Thr) is a strong determinant for the recognition by ThrRS. (+info)
Posttranscriptional modification of transfer RNA in the submarine hyperthermophile Pyrolobus fumarii.
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In the RNA of hyperthermophiles, which grow optimally between 80 degrees C and 106 degrees C, posttranscriptional modification has been identified as a leading mechanism of structural stabilization. Particularly in the Archaeal evolutionary domain these modifications are expressed as a structurally diverse array of modification motifs, many of which include ribose methylation. Using mass spectrometric techniques we have examined the posttranscriptional modifications in unfractionated tRNA from the remarkable organism Pyrolobus fumarii, which grows optimally at 106 degrees C, but up to 113 degrees C (Blochl et al. (1997), Extremophiles, 1, 14-21). Twenty-six modified nucleosides were detected, 11 of which are methylated in ribose. A new RNA nucleoside, 1,2'-O-dimethylguanosine (m1Gm) was characterized and the structure confirmed by chemical synthesis. (+info)
Taxonomic problems arising in the genera Haloterrigena and Natrinema.
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Data currently available on members of the genera Haloterrigena and Natrinema suggest that some strains of the species Haloterrigena turkmenica that have been shown by DNA-DNA hybridization to belong to this species show a high degree of 16S rDNA sequence similarity to members of the genus NATRINEMA: However, closer examination of the data presented in the original publications and in subsequent publications suggests that there may be a number of problems associated with some of the published data. (+info)