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Crystallization of the RNA‐binding domain of the transcriptional antiterminator protein sacy from Bacillus subtilis
Author(s) -
Manival Xavier,
Aymerich Stéphane,
Strub MariePaule,
Dumas Christian,
Kochoyan Michel,
van Tilbeurgh Herman
Publication year - 1997
Publication title -
proteins: structure, function, and bioinformatics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.699
H-Index - 191
eISSN - 1097-0134
pISSN - 0887-3585
DOI - 10.1002/(sici)1097-0134(199708)28:4<590::aid-prot13>3.0.co;2-b
Subject(s) - bacillus subtilis , terminator (solar) , antitermination , rna binding protein , rna , biology , crystallization , transcription (linguistics) , binding domain , biochemistry , microbiology and biotechnology , chemistry , biophysics , binding site , rna polymerase , gene , genetics , physics , ionosphere , organic chemistry , astronomy , bacteria , linguistics , philosophy
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 Å). 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. Proteins 28:590–594, 1997. © 1997 Wiley‐Liss, Inc.