Crystallisation of RNA-protein complexes. II. The application of protein engineering for crystallisation of the U1A protein-RNA complex.
The hairpin is one of the most commonly found structural motifs of RNA and is often a binding site for proteins. Crystallisation of U1A spliceosomal protein bound to a RNA hairpin, its natural binding site on U1snRNA, is described. RNA oligonucleotides were synthesised either chemically or by in vitro transcription using T7 RNA polymerase and purified to homogeneity by gel electrophoresis. Crystallisation trials with the wild-type protein sequence and RNA hairpins containing various stem sequences and overhanging nucleotides only resulted in a cubic crystal form which diffracted to 7-8 A resolution. A new crystal form was grown by using a protein variant containing mutations of two surface residues. The N-terminal sequence of the protein was also varied to reduce heterogeneity which was detected by protein mass spectrometry. A further crystallisation search using the double mutant protein and varying the RNA hairpins resulted in crystals diffracting to beyond 1.7 A. The methods and strategy described in this paper may be applicable to crystallisation of other RNA-protein complexes.
Oubridge
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C
MRC Laboratory of Molecular Biology, Cambridge, UK.
Ito
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N
Teo
C H
CH
Fearnley
I
I
Nagai
K
K
D000595:Amino Acid Sequence; D001483:Base Sequence; D003196:Computer Graphics; D003198:Computer Simulation; D003460:Crystallization; D018360:Crystallography, X-Ray; D003545:Cysteine; D012321:DNA-Directed RNA Polymerases; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D015202:Protein Engineering / Q000379:methods*; D012342:RNA, Small Nuclear / Q000138:chemical synthesis / Q000737:chemistry* / Q000302:isolation & purification*; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D017412:Ribonucleoprotein, U1 Small Nuclear / Q000737:chemistry* / Q000302:isolation & purification*; D014158:Transcription, Genetic; D014764:Viral Proteins
D012342:RNA, Small Nuclear; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D017412:Ribonucleoprotein, U1 Small Nuclear; C075339:U1A protein; D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D003545:Cysteine