Mechanism Of Transcription Regulation And Stability Of The Quorum Sensing Protein Trar

Abstract

TraR is a transcription activator of the quorum sensing system in the plant pathogen Agrobacterium tumefaciens. This protein is composed of two domains: amino-terminal domain (NTD) which is responsible for binding the autoinducer OOHL and for dimerization and the carboxyl-terminal domain (CTD) which contributes less extensively to dimerization and binds to specific sequences in the DNA called tra boxes. Despite being one of the most studied proteins from the LuxR family, the mechanism of transcription activation and stability of TraR are not well understood. In the first part of this work, we construct mutations in amino acids in the NTD of TraR to find the ones involved in the contact with RNA polymerase. Two patches of amino acids were found. One of them is composed of amino acids that are important for class I and II promoters. Therefore, those amino acids might contact the alpha-CTD subunit of the RNA polymerase. The other patch has amino acids important only for class II promoters, indicating that they might contact alpha or sigma subunits of the RNA polymerase. The second part of this study shows that the CTD of TraR contains amino acids that destabilize the protein. The results indicate that amino acids that are exposed in monomer TraR, but buried in the dimer protein, can provide protease recognition motifs. It was also shown that amino acids involved in RNA polymerase and DNA contacts, can also destabilize TraR. These results suggest that when bound to DNA or RNA polymerase, this amino acids would not be available for proteolysis. In the third part of this study, the effect of the antiactivator protein, TraM, in the stability of TraR was studied. The results show that TraR is degraded after antiactivation and that the levels of TraM do not change. Mutants that block antiactivation also block TraR proteolysis. The results suggest that TraM is an adaptor protein which delivers TraR to proteases, increasing its degradation rate.