STRUCTURE-FUNCTION ANALYSIS OF THE RAT PROLACTIN PROMOTER - PHASING REQUIREMENTS OF PROXIMAL CELL-SPECIFIC ELEMENTS

Expression of PRL, a member of the GH family of genes, is restricted to the lactotroph cells of the anterior pituitary. The proximal promoter of the rat PRL (rPRL) gene contains four factor-binding sites. Three nonadjacent elements, footprints (FP) I, III, and IV, are separated by an integral number of helical turns and bind a pituitary-specific factor, LSF-1. FP II binds another factor present in pituitary and nonpituitary cells. The mechanisms by which DNA-bound proteins influence RNA polymerase-II activity over large distances are not fully understood, but protein-protein interactions, with looping of intervening DNA, may bring distant sites into close proximity. Here, we demonstrate, using protein titration studies, that LSF-1 binds to the most proximal FP I element with the highest affinity, whereas it binds the more distal elements, FP III and FP IV, with progressively lower affinities. Time-course and salt-sensitivity studies reveal that binding of LSF-1 to all three pituitary-specific rPRL promoter sites occurs rapidly (less-than-or-equal-to 1 min) and requires fairly high salt concentrations (greater-than-or-equal-to 300 mM KCI) to destabilize protein-DNA interactions. Moreover, once bound, the pituitary nuclear factor(s) induces a conformational change in rPRL DNA structure with greatly delayed kinetics (> 15 min) and at a different salt concentration than are required for simply factor binding. Taken together, these data suggest a model in which LSF-1 initially binds fairly rapidly to multiple nonadjacent elements and then interacts with itself or other DNA-bound proteins much more slowly, possibly looping or bending the rPRL promoter. To test the functional significance of this model directly, we constructed phasing mutants of the rPRL promoter with insertions or deletions between the proximal and distal footprints. The results show that in vitro transcription rates are markedly reduced when these elements are on opposite faces of the DNA. Therefore, these data provide novel insights into the molecular mechanisms of rPRL gene control and imply that factor-induced alterations in promoter structure, via protein-protein interactions, may be critical in stabilizing a productive transcription initiation complex.