Specificity of the interaction of RocR with the rocG-rocA intergenic region in Bacillus subtilis.
In Bacillus subtilis, expression of the rocG gene, encoding glutamate dehydrogenase, and the rocABC operon, involved in arginine catabolism, requires SigL (sigma(54))-containing RNA polymerase as well as RocR, a positive regulator of the NtrC/NifA family. The RocR protein was purified and shown to bind specifically to the intergenic region located between rocG and the rocABC operon. DNaseI footprinting experiments were used to define the RocR-binding site as an 8 bp inverted repeat, separated by one base pair, forming an imperfect palindrome which is present twice within the rocG-rocABC intergenic region, acting as both a downstream activating sequence (DAS) and an upstream activating sequence (UAS). Point mutations in either of these two sequences significantly lowered expression of both rocG and rocABC. This bidirectional enhancer element retained partial activity even when moved 9 kb downstream of the rocA promoter. Electron microscopy experiments indicated that an intrinsically curved region is located between the UAS/DAS region and the promoter of the rocABC operon. This curvature could facilitate interaction of RocR with sigma(54)-RNA polymerase at the rocABC promoter.
Ali
Naima Ould
NO
Unité de Biochimie Microbienne, Institut Pasteur, URA 2172 du Centre National de la Recherche Scientifique, 25 rue du Docteur Roux, 75724 Paris Cedex 15, France.
Jeusset
Josette
J
Laboratoire de Microscopie Cellulaire et Moléculaire, Institut Gustave Roussy, UMR 1598 du Centre National de la Recherche Scientifique, 94805 Villejuif Cedex, France.
Larquet
Eric
E
Laboratoire de Minéralogie Cristallographie, Université Paris 6, UMR 7590, IPGP, CNRS Case 115, Tour 16, 4 Place Jussieu, 75252 Paris Cedex 05, France.|Groupe de Microscopie Structurale Moléculaire, Institut Pasteur, URA 2185 du Centre National de la Recherche Scientifique, 25 rue du Docteur Roux, 75724 Paris Cedex 15, France.
Le Cam
Eric
E
Laboratoire de Microscopie Cellulaire et Moléculaire, Institut Gustave Roussy, UMR 1598 du Centre National de la Recherche Scientifique, 94805 Villejuif Cedex, France.
Belitsky
Boris
B
Department of Molecular Biology and Microbiology, Tufts University, School of Medicine, Boston, MA 02111, USA.
Sonenshein
Abraham L
AL
Department of Molecular Biology and Microbiology, Tufts University, School of Medicine, Boston, MA 02111, USA.
Msadek
Tarek
T
Unité de Biochimie Microbienne, Institut Pasteur, URA 2172 du Centre National de la Recherche Scientifique, 25 rue du Docteur Roux, 75724 Paris Cedex 15, France.
Débarbouillé
Michel
M
Unité de Biochimie Microbienne, Institut Pasteur, URA 2172 du Centre National de la Recherche Scientifique, 25 rue du Docteur Roux, 75724 Paris Cedex 15, France.
D001120:Arginine / Q000378:metabolism; D001412:Bacillus subtilis / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D018983:DNA Footprinting; D021901:DNA, Intergenic / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003851:Deoxyribonucleases / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D005969:Glutamate Dehydrogenase / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D009876:Operon; D015534:Trans-Activators / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D021901:DNA, Intergenic; D015534:Trans-Activators; C086032:rocR protein, Bacillus subtilis; D001120:Arginine; D005969:Glutamate Dehydrogenase; D003851:Deoxyribonucleases