Fig. 6. Identification of the pilA transcription start site. (A) Sequence of the pilA promoter region. The pilA transcription start site is marked with a +1 and an arrow. Three regions which are protected by CtrA∼P (see Figure 7) are underlined, and CtrA binding motifs are marked in bold. A divergently transcribed gene, orfX, is 316 nt upstream of the predicted PilA methionine. (B) Start site of pilA transcription determined by primer extension. Ten micrograms of total RNA obtained from NA1000 or 10 µg of yeast tRNA were hybridized with the pilinrev2 primer and transcribed with Superscript II at 42°C. A sequence ladder generated with the same primer was used to determine the start site of transcription. The sequencing ladder corresponds to the non-coding strand. The doublet start site indicated with small arrows, and designated in bold, was observed with several different RNA preparations and two additional primers (data not shown).