Identification and characterization of a ran gene promoter in the protozoan pathogen Giardia lamblia

The promoter elements that regulate transcription initiation in Giardia lam blia are poorly understood. In this report, the promoter of the Giardia ran gene was studied using a luciferase expression plasmid pRANluc+ to monitor transcription efficiency. An AT-rich sequence spanning -51/-20 relative to the translation start site of the ran gene was identified and was found to be required for efficient luciferase expression by deletion and mutation m apping of pRANluc+. The -51/-20 sequence was also sufficient for promoter a ctivity as revealed from studies on a 32-base pair synthetic promoter deriv ed from this region. Deletion mapping of the synthetic promoter revealed tw o minimal promoter elements, -51/-42 and -30/-20, sufficient for 6- and 30- fold luciferase expression above background, respectively. The transcriptio n start sites on luc+ messenger RNA were determined by the position of the synthetic promoter in the luciferase expression plasmids as shown by primer extension experiments. Results from electrophoretic mobility shift assays revealed multiple DNA-protein complexes upon binding of nuclear proteins wi th either DNA strand but not the double-stranded DNA derived from the ran p romoter. Our results delineate the first promoter sequence of the Giardia g ene (ran), which provides an excellent model for future studies on transcri ption regulation in this protozoan parasite.