CRYSTALLIZATION OF THE RNA-BINDING DOMAIN OF THE TRANSCRIPTIONAL ANTITERMINATOR PROTEIN SACY FRONT BACILLUS-SUBTILIS

SacY is the antiterminator protein involved in the induction by sucros e of the expression of the levansucrase gene (sacB) of Bacillus subtil is. In the presence of sucrose, SacY is activated and prevents prematu re termination of transcription by binding to a RNA-antiterminator (RA T) sequence partially overlapping with the terminator sequence. SacY c onsists of a RNA-binding N-terminal domain, SacY(1-55), and a regulato ry domain, SacY(57-280), sensitive to the sucrose concentration. SacY( 1-55) is in itself capable of binding to the RAT sequence and preventi ng termination independently of the sucrose concentration. In this pap er we describe the overexpression. In this paper we describe the overe xpression, the purification, and the crystallization of SacY(1-55). We obtained six different crystal forms, some of them diffracting to hig h resolution (> 1.5 Angstrom). Self rotation function calculations ind icated the presence of a dimer in the asymmetric unit, which is in agr eement with a proposed oligomeric state in solution as observed by hig h-resolution NMR measurements. The crystallization of some site-direct ed cysteine mutants opens the way of solving the structure by multiple isomorphous replacement. (C) 1997 Wiley-Liss, Inc.