Crystallization of RNA-protein complexes I. Methods for the large-scale preparation of RNA suitable for crystallographic studies
Identifieur interne : 004199 ( Main/Exploration ); précédent : 004198; suivant : 004200Crystallization of RNA-protein complexes I. Methods for the large-scale preparation of RNA suitable for crystallographic studies
Auteurs : Stephen R. Price [Royaume-Uni] ; Nobutoshi Ito [Royaume-Uni] ; Chris Oubridge [Royaume-Uni] ; Johanna M. Avis [Royaume-Uni] ; Kiyoshi Nagai [Royaume-Uni]Source :
- Journal of Molecular Biology [ 0022-2836 ] ; 1995.
English descriptors
- KwdEn :
- Teeft :
- Alkaline phosphatase, Autolytic processing, Binding proteins, Biochemical studies, Bsmai, Bsmai restriction site, Buzayan, Chemical synthesis, Cleavage, Cleavage reaction, Cleavage site, Cleavage sites, Cognate, Complex structures, Comprehensive review, Control incubation, Crystal structure, Crystallisation, Crystallographic studies, Cyclic, Cyclic phosphate, Denaturing polyacrylamide, Double hammerhead ribozyme, Double ribozyme method, Efficient cleavage, Final concentration, Further characterisation, Gerlach, Hairpin, Hairpin ribozyme, Hammerhead, Hammerhead ribozyme, Hammerhead ribozymes, Hampel, Haseloff gerlach, Kinase, Large amount, Large scale, Large scale preparation, Linearised, Linearised plasmid, Linearised plasmid template, Lower band, Maleic acid, Molecular biology, Natl acad, Nucl, Nucleotide, Nucleotide sequence, Phage polynucleotide kinase, Phosphatase, Phosphatase activity, Phosphorothioate, Phosphorylation reaction, Plasmid, Plasmid template, Polymerase, Polynucleotide, Polynucleotide kinase, Preparative scale, Promoter sequence, Restriction site, Ribozyme, Ribozymes, Rna, Satellite tobacco ringspot virus, Secondary structure, Sequence requirement, Sequence requirements, Sequence restriction, Sequence restrictions, Sheldon symons, Show reaction, Structural studies, Subsequent incubation, Terminus, Third method, Transcription, Transcription product, Transcription products, Transcription reactions, Uhlenbeck, Ultraviolet shadowing, Upper band.
Abstract
In vitro transcription using bacteriophage RNA polymerases and linearised plasmid or oligodeoxynucleotide templates has been used extensively to produce RNA for biochemical studies. This method is, however, not ideal for generating RNA for crystallisation because efficient synthesis requires the RNA to have a purine rich sequence at the 5′ terminus, also the subsequent RNA is heterogenous in length. We have developed two methods for the large scale production of homogeneous RNA of virtually any sequence for crystallization. In the first method RNA is transcribed together with two flanking intramolecularly-, (cis-), acting ribozymes which excise the desired RNA sequence from the primary transcript, eliminating the promoter sequence and heterogeneous 3′ end generated by run-off transcription. We use a combination of two hammerhead ribozymes or a hammerhead and a hairpin ribozyme. The RNA-enzyme activity generates few sequence restrictions at the 3′ terminus and none at the 5′ terminus, a considerable improvement on current methodologies. In the second method the Bsm AI restriction endonuclease is used to linearize plasmid template DNA thereby allowing the generation of RNA with any 3′ end. In combination with a 5′ cis-acting hammerhead ribozyme any sequence of RNA may be generated by in vitro transcription. This has proven to be extremely useful for the synthesis of short RNAs.
Url:
DOI: 10.1006/jmbi.1995.0305
Affiliations:
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Avis</name></author><author><name sortKey="Nagai, Kiyoshi" sort="Nagai, Kiyoshi" uniqKey="Nagai K" first="Kiyoshi" last="Nagai">Kiyoshi Nagai</name></author></titleStmt><publicationStmt><idno type="wicri:source">ISTEX</idno><idno type="RBID">ISTEX:24E4BEF54EC7D6CA848A7E94FFED99711D27237D</idno><date when="1995" year="1995">1995</date><idno type="doi">10.1006/jmbi.1995.0305</idno><idno type="url">https://api.istex.fr/ark:/67375/6H6-DJMGVD6R-N/fulltext.pdf</idno><idno type="wicri:Area/Istex/Corpus">000E13</idno><idno type="wicri:explorRef" wicri:stream="Istex" wicri:step="Corpus" wicri:corpus="ISTEX">000E13</idno><idno type="wicri:Area/Istex/Curation">000E13</idno><idno type="wicri:Area/Istex/Checkpoint">001890</idno><idno type="wicri:explorRef" wicri:stream="Istex" wicri:step="Checkpoint">001890</idno><idno type="wicri:doubleKey">0022-2836:1995:Price S:crystallization:of:rna</idno><idno type="wicri:Area/Main/Merge">004260</idno><idno type="wicri:Area/Main/Curation">004199</idno><idno type="wicri:Area/Main/Exploration">004199</idno></publicationStmt><sourceDesc><biblStruct><analytic><title level="a" type="main" xml:lang="en">Crystallization of RNA-protein complexes I. 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This method is, however, not ideal for generating RNA for crystallisation because efficient synthesis requires the RNA to have a purine rich sequence at the 5′ terminus, also the subsequent RNA is heterogenous in length. We have developed two methods for the large scale production of homogeneous RNA of virtually any sequence for crystallization. In the first method RNA is transcribed together with two flanking intramolecularly-, (cis-), acting ribozymes which excise the desired RNA sequence from the primary transcript, eliminating the promoter sequence and heterogeneous 3′ end generated by run-off transcription. We use a combination of two hammerhead ribozymes or a hammerhead and a hairpin ribozyme. The RNA-enzyme activity generates few sequence restrictions at the 3′ terminus and none at the 5′ terminus, a considerable improvement on current methodologies. In the second method the Bsm AI restriction endonuclease is used to linearize plasmid template DNA thereby allowing the generation of RNA with any 3′ end. In combination with a 5′ cis-acting hammerhead ribozyme any sequence of RNA may be generated by in vitro transcription. This has proven to be extremely useful for the synthesis of short RNAs.</div></front></TEI><affiliations><list><country><li>Royaume-Uni</li></country></list><tree><country name="Royaume-Uni"><noRegion><name sortKey="Price, Stephen R" sort="Price, Stephen R" uniqKey="Price S" first="Stephen R." last="Price">Stephen R. Price</name></noRegion><name sortKey="Avis, Johanna M" sort="Avis, Johanna M" uniqKey="Avis J" first="Johanna M." last="Avis">Johanna M. Avis</name><name sortKey="Ito, Nobutoshi" sort="Ito, Nobutoshi" uniqKey="Ito N" first="Nobutoshi" last="Ito">Nobutoshi Ito</name><name sortKey="Nagai, Kiyoshi" sort="Nagai, Kiyoshi" uniqKey="Nagai K" first="Kiyoshi" last="Nagai">Kiyoshi Nagai</name><name sortKey="Oubridge, Chris" sort="Oubridge, Chris" uniqKey="Oubridge C" first="Chris" last="Oubridge">Chris Oubridge</name></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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