The process of Agrobacterium tumefaciens of plants is a well studied phenomenon, and is the only bonafide example of natural genetic engineering. The infecting bacterium transfers a fragment of its DNA from a tumor inducing (Ti) plasmid, into the host plant genome. Genes encoded on this fragment direct tumor formation and the synthesis of unusual sugars and amino acids termed opines, which serve as Agrobacterium-specific carbon/nitrogen sources. In turn, the opines stimulate the expression of A. tumefaciens genes involved in their uptake and utilization, conjugal transfer of the Ti plasmid, and a number of other functions that may be involved in tumor colonization. In octopine-type plasmids this induction is mediated at least in part by the LysR-type transcriptional activator OccR, which induces the transcription of these genes in response to the opine octopine and which also autoregulates its own synthesis. One of the targets for OccR regulation is the overlapping, divergently transcribed promoter region between the occ (octopine catabolism) operon and the occR gene itself. This unusual genetic organization is typical of LysR-type systems and probably allows simultaneous control of both operons. In the proposed study, we shall examine the interaction of OccR with this transcriptional control region. Evidence is presented that OccR binds to this region in the presence or absence of octopine, and that octopine induces conformational changes in Occr and/or its target site. We hypothesize that; i) OccR bends its target site, that octopine incites modulation of this bending, and that these changes result in the induction of transcription, or ii) that octopine-induced conformational changes in OccR itself result in transcriptional activation. The proposed investigation will employ biochemical, molecular biological, and genetic techniques to test these hypotheses.
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