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At least 1,225 records · Page 68Linked to original sources
PubMed2003

In vitro RNP assembly and methylation guide activity of an unusual box C/D RNA, cis-acting archaeal pre-tRNA(Trp).

Among the large family of C/D methylation guide RNAs, the intron of euryarchaeal pre-tRNA(Trp) represents an outstanding specimen able to guide in cis, instead of in trans, two 2'-O-methylations in the pre-tRNA exons. Remarkably, both sites of methylation involve nucleotides within the bulge-helix-bulge (BHB) splicing motif, while the RNA-guided methylation and pre-tRNA splicing events depend on mutually exclusive RNA folding patterns. Using the three recombinant core proteins of archaeal C/D RNPs, we have analyzed in vitro RNP assembly of the pre-tRNA and tested its site-specific methylation activity. Recognition by L7Ae of hallmark K-turns at the C/D and C'/D' motifs appears as a crucial assembly step required for subsequent binding of a Nop5p-aFib heterodimer at each site. Unexpectedly, however, even without L7Ae but at a higher concentration of Nop5p-aFib, a substantially active RNP complex can still form, possibly reflecting the higher propensity of the cis-acting system to form guide RNA duplex(es) relative to classical trans- acting C/D RNA guides. Moreover, footprinting data of RNPs, consistent with Nop5p interacting with the non-canonical stem of the K-turn, suggest that binding of Nop5p-aFib to the pre-tRNA-L7Ae complex might direct transition from a splicing-competent structure to an RNA conformer displaying the guide RNA duplexes required for site-specific methylation.

Archaeal Proteins↗
PubMed2006

THUMP from archaeal tRNA:m22G10 methyltransferase, a genuine autonomously folding domain.

The tRNA:m2(2)G10 methyltransferase of Pyrococus abyssi (PAB1283, a member of COG1041) catalyzes the N2,N2-dimethylation of guanosine at position 10 in tRNA. Boundaries of its THUMP (THioUridine synthases, RNA Methyltransferases and Pseudo-uridine synthases)--containing N-terminal domain [1-152] and C-terminal catalytic domain [157-329] were assessed by trypsin limited proteolysis. An inter-domain flexible region of at least six residues was revealed. The N-terminal domain was then produced as a standalone protein (THUMPalpha) and further characterized. This autonomously folded unit exhibits very low affinity for tRNA. Using protein fold-recognition (FR) methods, we identified the similarity between THUMPalpha and a putative RNA-recognition module observed in the crystal structure of another THUMP-containing protein (ThiI thiolase of Bacillus anthracis). A comparative model of THUMPalpha structure was generated, which fulfills experimentally defined restraints, i.e. chemical modification of surface exposed residues assessed by mass spectrometry, and identification of an intramolecular disulfide bridge. A model of the whole PAB1283 enzyme docked onto its tRNA(Asp) substrate suggests that the THUMP module specifically takes support on the co-axially stacked helices of T-arm and acceptor stem of tRNA and, together with the catalytic domain, screw-clamp structured tRNA. We propose that this mode of interactions may be common to other THUMP-containing enzymes that specifically modify nucleotides in the 3D-core of tRNA.

Amino Acid Sequence↗
PubMed2004

Analyses of PCR products using DNA templates containing a consecutive deoxyinosine sequence.

We determined nucleotide incorporation specificity of four thermostable DNA polymerases opposite deoxyinosine in order to evaluate the mutagenic effect of the presence of a consecutive deoxyinosine sequence during PCR amplification. Deoxycytosine was the only incorporated nucleotide opposite deoxyinosine during PCR amplification, irrespective of origin of DNA polymerase.

Base Pairing↗
PubMed2005

RNA-guided RNA modification: functional organization of the archaeal H/ACA RNP.

In eukaryotes and archaea, uridines in various RNAs are converted to pseudouridines by RNA-guided RNA modification complexes termed H/ACA RNPs. Guide RNAs within the complexes base-pair with target RNAs to direct modification of specific ribonucleotides. Cbf5, a protein component of the complex, likely catalyzes the modification. However, little is known about the organization of H/ACA RNPs and the roles of the multiple proteins thought to comprise the complexes. We have reconstituted functional archaeal H/ACA RNPs from recombinant components, defined the components necessary and sufficient for function, and determined the direct RNA-protein and protein-protein interactions that occur between the components. The results provide substantial insight into the functional organization of this RNP. The functional complex requires a guide RNA and each of four proteins: Cbf5, Gar1, L7Ae, and Nop10. Two proteins interact directly with the guide RNA: L7Ae and Cbf5. L7Ae does not interact with other H/ACA RNP proteins in the absence of the RNA. We have defined two novel functions for Cbf5. Cbf5 is the protein that specifically recognizes and binds H/ACA guide RNAs. In addition, Cbf5 recruits the two other essential proteins, Gar1 and Nop10, to the pseudouridylation guide complex.

Archaeal Proteins↗
PubMed2002

Semi-automatic protein crystallization system that allows in situ observation of X-ray diffraction from crystals in the drop.

Semi-automatic protein crystallization device was developed using 96 format Hydra dispenser. Eight extra syringes were added for pipetting protein solution with its own dispensing mechanism. This device allows setting up 96 drops in about three minutes. New crystallization plate has also been developed. The plate allows collecting an oscillation photograph of the crystal directly in the drop. The diffraction quality of the crystals in the drops can be inspected without causing any other physical perturbations to the crystals. With the modified dispenser and this plate, we can perform both hanging and sitting drop vapour diffusion method.

Archaeal Proteins↗
PubMed2006

Combining experimental data for structure determination of flexible multimeric macromolecules by molecular replacement.

A major effort has been made by the structural biology community to develop user-friendly software for the use of biologists. However, structural projects become more and more challenging and their solution often relies on a combination of information from various sources. Here, it is described how X-ray data, normal-mode analysis (NMA) and electron-microscopy (EM) data can be successfully combined in order to obtain a molecular-replacement (MR) solution for crystal structures containing multimeric molecules. NMA is used to simulate computationally the inherent internal flexibility of the monomer and thus enhance, together with the crystal noncrystallographic symmetry (NCS), the MR capabilities. NCS is also used to obtain a reliable EM reconstruction, which is then employed as a filter to construct oligomers starting from monomers. The feasibility of the direct use of EM reconstructions as a template for MR when the X-ray and EM data resolutions overlap is also discussed.

Agrobacterium tumefaciens↗
PubMed1999

Photodegradation of organic waste coupling hydrogenase and titanium dioxide.

In this work the results of the study on the degradation of dairy waste and lactose with use of titanium dioxide (TiO2), as photocatalyst, are presented. The ability of TiO2 to degrade dairy waste is compared either in the presence of molecular oxygen or of a bacterial hydrogenase as electron acceptor. The enzyme-mediated H2 production rates or the O2 consumption rates are used to measure electron donor degradation. The results obtained clearly indicate that dairy waste can be degraded by the TiO2 photocatalyst. Proteins present in the dairy waste have a strong inhibitory effect on the degradation process. Indeed lactose alone can easily be degraded. When the protein extract obtained from the dairy waste was added to the lactose solution, the reaction for both electron acceptors, hydrogenase and oxygen, was inhibited. When the dairy waste solutions were diluted, there was a positive effect on the reaction rate. This was particularly true in the case of hydrogenase, and to a lesser extent in the case of oxygen acceptor. The reduction of the pH from 8 to 6 also increased H2 production when the enzyme was used.

Chromatography, Gas↗
PubMed2002

An alternative flavin-dependent mechanism for thymidylate synthesis.

Although deoxythymidylate cannot be provided directly by ribonucleotide reductase, the gene encoding thymidylate synthase ThyA is absent from the genomes of a large number of nonsymbiotic microbes. We show that ThyX (Thy1) proteins of previously unknown function form a large and distinct class of thymidylate synthases. ThyX has a wide but sporadic phylogenetic distribution, almost exclusively limited to microbial genomes lacking thyA. ThyX and ThyA use different reductive mechanisms, because ThyX activity is dependent on reduced flavin nucleotides. Our findings reveal complexity in the evolution of thymidine in present-day DNA. Because ThyX proteins are found in many pathogenic microbes, they present a previously uncharacterized target for antimicrobial compounds.

Catalysis↗
PubMed1999

Occurrence of free D-amino acids and aspartate racemases in hyperthermophilic archaea.

The occurrence of free D-amino acids and aspartate racemases in several hyperthermophilic archaea was investigated. Aspartic acid in all the hyperthermophilic archaea was highly racemized. The ratio of D-aspartic acid to total aspartic acid was in the range of 43.0 to 49.1%. The crude extracts of the hyperthermophiles exhibited aspartate racemase activity at 70 degrees C, and aspartate racemase homologous genes in them were identified by PCR. D-Enantiomers of other amino acids (alanine, leucine, phenylalanine, and lysine) in Thermococcus strains were also detected. Some of them might be by-products of aspartate racemase. It is proven that D-amino acids are produced in some hyperthermophilic archaea, although their function is unknown.

Amino Acid Isomerases↗
PubMed2001

Isolation of PDX2, a second novel gene in the pyridoxine biosynthesis pathway of eukaryotes, archaebacteria, and a subset of eubacteria.

In this paper we describe the isolation of a second gene in the newly identified pyridoxine biosynthesis pathway of archaebacteria, some eubacteria, fungi, and plants. Although pyridoxine biosynthesis has been thoroughly examined in Escherichia coli, recent characterization of the Cercospora nicotianae biosynthesis gene PDX1 led to the discovery that most organisms contain a pyridoxine synthesis gene not found in E. coli. PDX2 was isolated by a degenerate primer strategy based on conserved sequences of a gene specific to PDX1-containing organisms. The role of PDX2 in pyridoxine biosynthesis was confirmed by complementation of two C. nicotianae pyridoxine auxotrophs not mutant in PDX1. Also, targeted gene replacement of PDX2 in C. nicotianae results in pyridoxine auxotrophy. Comparable to PDX1, PDX2 homologues are not found in any of the organisms with homologues to the E. coli pyridoxine genes, but are found in the same archaebacteria, eubacteria, fungi, and plants that contain PDX1 homologues. PDX2 proteins are less well conserved than their PDX1 counterparts but contain several protein motifs that are conserved throughout all PDX2 proteins.

Amino Acid Sequence↗
PubMed2003

RSEARCH: finding homologs of single structured RNA sequences.

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.

Animals↗
PubMed2002

Cloning, expression, and characterization of thermostable DNA polymerase from Thermoanaerobacter yonseiensis.

A gene, coined tay, for a thermostable DNA polymerase from the novel, extremely thermophilic bacterium Thermoanaerobacter yonseiensis was cloned and expressed in E. coli. Using a DNA polymerase homologous PCR product as a hybridization probe, tay was isolated and sequenced to consist of 2,616 nucleotides that encode 872 amino acids. A database analysis showed that DNA polymerase, coined Tay, from T. yonseiensis shared a 39 percent to 47 percent identity in the amino acid sequence with those from other DNA polymerases. Tay was overexpressed in E. coli as a fusion protein with a poly-histidine tag at the Cterminus. It was purified by heat treatment, followed by a Ni(2+)-chelate column. The molecular weight of purified Tay was approximately 97 kDa, as shown by SDS PAGE, and it showed high DNA polymerase activity and thermostability. However, it had no 3'-->5' exonuclease activity

Amino Acid Sequence↗