FIG. 3.

Primer extension and 5′ RACE analyses of the pipX gene promoter region. (A) Primer extension analysis was carried out with primer PX18 and with RNAs isolated from bubbled cultures of Anabaena sp. strain PCC 7120, strain CSE2 (ntcA), and strain 216 (hetR) grown with ammonium (lanes 0) and incubated in the absence of combined nitrogen for the times indicated (in hours). The positions of the −436 and −388 RNA 5′ ends are indicated. (B) Primer extension analysis carried out with primer asr0485-7120-1 and RNA isolated from bubbled cultures of Anabaena sp. strain PCC 7120 grown with ammonium (lanes 0) and incubated in the absence of combined nitrogen for the times indicated (in hours). The positions of the −193 and −107 5′ RNA ends are indicated. A sequence ladder of the same DNA region is shown at the left. (C) 5′ RACE analysis was carried out with RNA isolated from bubbled cultures of Anabaena sp. strain PCC 7120 grown with ammonium (0 h) and incubated in the absence of combined nitrogen (12 h) treated (+) or not (−) with TAP, as indicated, with PX17 as the gene-specific primer for the PCR step. The putative 5′ positions of some bands are indicated. Size standards (DNA molecular weight marker X; Roche) are shown in the left lane. (D) DNA sequences upstream from three identified pipX TSPs (see the text). The NtcA-protected region upstream of TSP −436 and putative −10 and −35 boxes are indicated. Conserved triplets of the NtcA binding site are boxed.