The carboxyl-terminal domain of TraR, a Streptomyces HutC family repressor, functions in oligomerization.
Efficient conjugative transfer of the Streptomyces plasmid pSN22 is accomplished by regulated expression of the tra operon genes, traA, traB, and spdB. The TraR protein is the central transcriptional repressor regulating the expression of the tra operon and itself and is classified as a member of the HutC subfamily in the helix-turn-helix (HTH) GntR protein family. Sequence information predicts that the N-terminal domain (NTD) of TraR, containing an HTH motif, functions in binding of DNA to the cis element; however, the function of the C-terminal region remains obscure, like that for many other GntR family proteins. Here we demonstrate the domain structure of the TraR protein and explain the role of the C-terminal domain (CTD). The TraR protein can be divided into two structural domains, the NTD of M1 to R95 and the CTD of Y96 to E246, revealed by limited proteolysis. Domain expression experiments revealed that both domains retained their function. An in vitro pull-down assay using recombinant TraR proteins revealed that TraR oligomerization depended on the CTD. A bacterial two-hybrid system interaction assay revealed that the minimum region necessary for this binding is R95 to P151. A mutant TraR protein in which Leu121 was replaced by His exhibited a loss of both oligomerization ability and repressor function. An in vitro cross-linking assay revealed preferential tetramer formation by TraR and the minimum CTD. These results indicate that the C-terminal R95-to-P151 region of TraR functions to form an oligomer, preferentially a tetramer, that is essential for the repressor function of TraR.
Kataoka
Masakazu
M
Department of Environmental Science and Technology, Faculty of Engineering, Shinshu University, Wakasato 4-17-1, Nagano-shi, Nagano 380-8553, Japan. mars@shinshu-u.ac.jp
Tanaka
Takeshi
T
Kohno
Toshiyuki
T
Kajiyama
Yusuke
Y
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019281:Dimerization; D008957:Models, Genetic; D017434:Protein Structure, Tertiary; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013302:Streptomyces / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D012097:Repressor Proteins