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Find video protocols related to scientific articles indexed in Pubmed.
GTP-dependent RNA 3-terminal phosphate cyclase from the hyperthermophilic archaeon Pyrococcus furiosus.
Genes Cells
PUBLISHED: 11-10-2011
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We discovered that the PF1549 gene in Pyrococcus furiosus encodes a very heat-stable RNA 3-terminal phosphate cyclase (Pf-Rtc). Although all previously reported Rtc proteins are ATP-dependent enzymes, we found that Pf-Rtc requires GTP for its cyclase activity at 95 °C. Low-level activation of the enzyme was also observed in the presence of dGTP but not other dNTPs, indicating that the guanine base is very important for Pf-Rtc activity. We analyzed a series of GTP analogues and found that the conversion from GTP to GMP is important for Pf-Rtc activity and that an excess of GMP inhibits this activity. Gel-shift analysis clearly showed that the RNA-binding activity of Pf-Rtc is totally dependent on the linear form of the 3-terminal phosphate, with an apparent K(d) value of 20 nm at 95°C. Furthermore, we found that Pf-Rtc may contribute to GTP-dependent RNA ligation activity through the PF0027 protein (a 2-5 RNA ligase-like protein in P. furiosus). The possible roles of Pf-Rtc and the importance of terminal phosphate structures in RNA are discussed.
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A novel three-unit tRNA splicing endonuclease found in ultrasmall Archaea possesses broad substrate specificity.
Nucleic Acids Res.
PUBLISHED: 08-31-2011
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tRNA splicing endonucleases, essential enzymes found in Archaea and Eukaryotes, are involved in the processing of pre-tRNA molecules. In Archaea, three types of splicing endonuclease [homotetrameric: ?(4), homodimeric: ?(2), and heterotetrameric: (??)(2)] have been identified, each representing different substrate specificity during the tRNA intron cleavage. Here, we discovered a fourth type of archaeal tRNA splicing endonuclease (?(2)) in the genome of the acidophilic archaeon Candidatus Micrarchaeum acidiphilum, referred to as ARMAN-2 and its closely related species, ARMAN-1. The enzyme consists of two duplicated catalytic units and one structural unit encoded on a single gene, representing a novel three-unit architecture. Homodimeric formation was confirmed by cross-linking assay, and site-directed mutagenesis determined that the conserved L10-pocket interaction between catalytic and structural unit is necessary for the assembly. A tRNA splicing assay reveal that ?(2) endonuclease cleaves both canonical and non-canonical bulge-helix-bulge motifs, similar to that of (??)(2) endonuclease. Unlike other ARMAN and Euryarchaeota, tRNAs found in ARMAN-2 are highly disrupted by introns at various positions, which again resemble the properties of archaeal species with (??)(2) endonuclease. Thus, the discovery of ?(2) endonuclease in an archaeon deeply branched within Euryarchaeota represents a new example of the coevolution of tRNA and their processing enzymes.
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What is Visualize?

JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

How does it work?

We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.

Video X seems to be unrelated to Abstract Y...

In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.