After the establishment of a crown gall tumor, a new class of host-released compounds must be perceived. These compounds include opines, which are synthesized by the transformed cells and utilized by the bacteria as nutrients. One such opine, octopine, is perceived by the OccR protein, a LysR-type protein that activates a 14 gene operon (Mol. Microbiol. 20: 1199). This operon contains octopine catabolic genes, two ABC-type permeases, and the traR gene (see below). OccR remains bound to its operator both in the presence and absence of octopine, but undergoes a conformational change in response to octopine. This protein causes a high-angle DNA bend at its binding site in the absence of octopine, while addition of octopine decreases the bend angle. Octopine also causes one or two OccR monomers to translocate one helical turn (see Cell 69: 659; J. Mol. Biol. 253: 32; and J. Mol. Biol. 253: 691).
Two diagrams describing an octopine-induced conformational change of bound OccR protein. Octopine (black dots) it thought to cause the promoter-proximal dimer of OccR to translocate by one helical turn, converting OccR from an inactive form to a form that is able to activate transcription. The first diagram emphasizes the ligand-responsive DNA bend, while the second diagram emphasizes the DNA sequences to which OccR may bind.
We are currently trying to understand the relationship between this OccR translocation and transcriptional activation. In the absence of inducer, the downstream OccR dimer binds on either side of the -35 region. This is incompatible with activation and may even inhibit expression. In the presence of octopine, the downstream OccR dimer occupies a site centered at -41, a position similar to the CRP protein at the gal promoter of E. coli. The upstream dimer remains locked at a unique "high affinity" site, which includes the sequence ATAAN7TTAT. We believe that the downstream dimer binds to a weakly similar site (ATTCN7TTCA). The similarity to the upstream site must be weak in order for OccR to be released from this site. In preliminary experiments, improving this downstream site to make it resemble the upstream site more closely appears to lock OccR into a single conformation.
We have identified all the genes required for octopine degradation. These include three Ti plasmid genes previously identified, as well at the arcAB and putA operons (see Figure). We are especially interested in transcription of putA, which is activated by the PutR protein. PutR is encoded by a gene that is transcribed divergently from putA. PutR is homologous to the Lrp protein of E. coli. PutR functions by mechanisms that are strikingly similar to those of OccR, even though the two are not homologous. PutR remains bound to its operator in the presence or absence of the inducer (proline), but undergoes an inducer-responsive conformational change that alters the DNase I footprint of the protein (see J. Bacteriol. 178:1872). PutR also causes a DNA bend at its binding site. Proline does not affect the magnitude of the bend angle but appears to alter the direction of this DNA bend. That is, proline untwists DNA at its binding site by approximately 70 degrees.
We have also used atomic force microscopy to image PutR-DNA images, and find that the two DNA ends that protrude from these complexes are highly bent. In addition, measurements of the end-to-end length of DNA in these complexes indicates that PutR condenses DNA into a globular nucleoprotein complex. (J. Mol. Biol. 288:811-824).