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

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 BsmAI 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.

Synthesis of HIV-1 Ψ-site RNA sequences with site specific incorporation of the fluorescent base analog 2-aminopurine

“…As an alternative to a hammerhead ribozyme, a hairpin ribozyme can be used at the 3′-end which has sequence requirements on the 5′ side of the cleavage site that are less stringent. 35 The 5′ DIS acceptor and 3′ truncated Ψ-site donor RNA sequence fragments prepared through chemical and enzymatic protocols, respectively, were separately purified before use in the ligation reaction. For each ligation reaction, optimal conditions were initially screened using a series of small 10 μl scale reactions, focusing on customizing reagent concentrations and buffer conditions previously reported to be the most critical for individual reaction yields.…”

A high-affinity interaction between NusA and the rrn nut site in Mycobacterium tuberculosis

“…7, was cloned as an EcoRI-XbaI fragment into pGEM3Zϩ. To generate homogeneous RNA for footprinting a plasmid, pRBZ1͞63, was made to obtain the M. tuberculosis nut site between a 5Ј hammerhead ribozyme and a 3Ј hepatitis ␦ ribozyme (25). pUC119␦V, a derivative of pUC18T7Pst␦V (26) containing a T7 promoter and a 3Ј hepatitis ␦ ribozyme, was used as vector to generate the self-cleaving construct, pRBZ1͞63.…”

A simple and efficient method to reduce nontemplated nucleotide addition at the 3′ terminus of RNAs transcribed by T7 RNA polymerase

“…The heterogeneity at the 39 ends of transcripts generated by polymerases in vitro can have detrimental effects in a number of applications+ In the production of infectious viral transcripts, 39 heterogeneity may reduce the synthesis by viral RNA replicase (Miller et al+, 1986;Sun et al+, 1996), or reduce the infectivity of the RNA (Murphy & Park, 1997)+ In biochemical and structural analyses of RNA structures, 39 heterogeneity may result in additional signals that complicate the interpretation of results and may prevent the effective crystallization of the RNA needed for X-ray crystallography (Price et al+, 1995)+ Previously, one commonly used strategy to generate a homogeneous RNA sample of less than 50 nt is to perform denaturing gel electrophoresis and to excise and elute the desired band (Wyatt et al+, 1991)+ For RNAs longer than 50 nt, where it is difficult to separate the Nϩ1 RNA from the desired RNA by gel electrophoresis, the transcript can be expressed with a cis-acting ribozyme sequence that will cleave at the desired positions (Dianzott & Bujarski, 1988;Price et al+, 1995), or by use of a mutant T7 RNA polymerase that is defective in its initiation rate (Gardner et al+, 1997)+ Both procedures require extensive effort that may be partially alleviated by the use of chemically synthesized DNA templates or PCRgenerated double-stranded templates containing ribose 29-methoxy moieties+ The quality and, often, the yield of the desired transcripts will be improved by this method+ The ability to generate less complex transcription products will permit more rapid RNA purification such as by high-pressure liquid chromatography+…”