Crystallization of the RNA-binding domain of the transcriptional antiterminator protein SacY from Bacillus subtilis.
SacY is the antiterminator protein involved in the induction by sucrose of the expression of the levansucrase gene (sacB) of Bacillus subtilis. In the presence of sucrose, SacY is activated and prevents premature termination of transcription by binding to a RNA-antiterminator (RAT) sequence partially overlapping with the terminator sequence. SacY consists of a RNA-binding N-terminal domain, SacY(1-55), and a regulatory domain, SacY(56-280), sensitive to the sucrose concentration. SacY(1-55) is in itself capable of binding to the RAT sequence and preventing termination independently of the sucrose concentration. In this paper we describe the overexpression, the purification, and the crystallization of SacY(1-55). We obtained six different crystal forms, some of them diffracting to high resolution (> 1.5 A). Self rotation function calculations indicated the presence of a dimer in the asymmetric unit, which is in agreement with a proposed oligomeric state in solution as observed by high-resolution NMR measurements. The crystallization of some site-directed cysteine mutants opens the way of solving the structure by multiple isomorphous replacement.
Manival
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Centre de Biochimie structurale, CNRS-U9950, INSERM-U414, Montpellier, France.
Aymerich
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Strub
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MP
Dumas
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Kochoyan
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van Tilbeurgh
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D001412:Bacillus subtilis / Q000737:chemistry*; D001426:Bacterial Proteins / Q000737:chemistry*; D001665:Binding Sites; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000378:metabolism; D012313:RNA / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D014157:Transcription Factors; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; C076894:SacY protein, Bacillus subtilis; D014157:Transcription Factors; C064872:antiterminator proteins, Bacteria; D012313:RNA