CHARACTERIZATION OF 2 PROTEIN ACTIVITIES THAT INTERACT AT THE PROMOTER OF THE TRYPANOSOMATID SPLICED LEADER RNA

All trypanosome mRNAs have a spliced leader (SL). The SL RNA gene in L eptomonas seymouri is a member of the small nuclear RNA gene family. H owever, the SL RNA is required in stoichiometric amounts for transspli cing during mRNA formation. Expression of the SL RNA gene requires seq uence elements at bp -60 to -70 and bp -30 to -40 upstream from the tr anscription initiation site. Using conventional and affinity chromatog raphy, we have identified and characterized an -122-kDa protein, promo ter-binding protein (PBP) -1, that binds to double-strand DNA. The PBP -1-binding site is within the bp -60 to -70 element determined by DNas e I footprinting. Therefore, PBP-1 is the first characterized double-s trand DNA binding activity that interacts with a trypanosome gene prom oter. A second protein, PBP-2, interacts with the PBP-1:DNA complex an d its DNase I footprint extends to include the second promoter element (bp -30 to -40). An alteration of the spacing between the two promote r elements or mutation of the second element decreases PBP-2/PBP-1:DNA stability. Taken together, these data suggest that PBP-1 and PBP-2 ar e components of a transcription initiation complex that assembles with in the SL RNA gene promoter.