Delineation of the prt promoter. (A) Plasmid reporter of prt promoter activity. The pho1 ORF was fused immediately downstream from a fragment of genomic DNA containing the prt transcription start site, a 20-nt 5′-UTR, and 633 nt of 5′ flanking prt DNA (presumed to include the prt promoter). Serial truncation of the upstream margins of the 5′ flanking prt DNA were made at positions −444, −110, −61, and −5 relative to the prt transcription start site. The nucleotide sequence from position −110 to +32 is shown below the cartoon depiction of the reporter. The pho1 AUG start codon is underlined. The prt transcription start site is indicated by the arrow. A putative TATA element is outlined by a box. A putative HomolD element is shaded gray. Two nucleobase changes shown above the sequence were introduced into the HomolD element in the context of the −633 reporter (B) Acid phosphatase activity of pho1Δ cells bearing the indicated reporter plasmids (LEU2). The −633* reporter plasmid contains the two nucleobase changes in the HomolD element. (C) Primer extension analysis of the prt-driven pho1 transcript was performed with total RNA isolated from pho1Δ cells bearing the indicated reporter plasmids (top panel). Primer extension analysis of actin mRNA from the same samples was performed as a control (bottom panel).
