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Crystallization of RNA-protein complexes I. Methods for the large-scale preparation of RNA suitable for crystallographic studies

Ito, N; Oubridge, C; Avis, JM; Nagai, K; Price, SR; (1995) Crystallization of RNA-protein complexes I. Methods for the large-scale preparation of RNA suitable for crystallographic studies. Journal of Molecular Biology , 249 (2) 398 - 408. 10.1006/jmbi.1995.0305. Green open access

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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.

Type: Article
Title: Crystallization of RNA-protein complexes I. Methods for the large-scale preparation of RNA suitable for crystallographic studies
Open access status: An open access version is available from UCL Discovery
DOI: 10.1006/jmbi.1995.0305
Publisher version: http://dx.doi.org/10.1006/jmbi.1995.0305
Language: English
Additional information: This article was published in the Journal of Molecular Biology, 249(2), Stephen R. Price, Nobutoshi Ito, Chris Oubridge, Johanna M. Avis, Kiyoshi Nagai, Crystallization of RNA-protein complexes I. Methods for the large-scale preparation of RNA suitable for crystallographic studies, 398-408, Copyright Elsevier 1995. Permission is granted to post the published papers as per the discretion of the Publishers. The Journal of Molecular Biology homepage can be found here: http://www.sciencedirect.com/science/journal/00222836.
Keywords: in vitro transcription; ribozymes; RNA-protein interactions; X-ray crystallography; oligoribonucleotide
UCL classification: UCL
UCL > Provost and Vice Provost Offices
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences > Cell and Developmental Biology
URI: https://discovery.ucl.ac.uk/id/eprint/76346
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