The relAP2 promoter contains a cAMP receptor protein
(CRP) binding site that is activated by CRP (Nakagawa
et al., 2006) and the relAP2(2100+50) reporter included
this CRP site. The microarray data also indicated that
crp mRNA levels were increased (1.9-fold) in cells
lacking 6S RNA compared with wild-type cells (A. T.Cavanagh & K. M. Wassarman, unpublished data). To test
whether changes in transcription at relAP2(2100+50) and
relAP1(2100+50) were likely to result from direct regulation
by 6S RNA or might be due to indirect regulation via
changes in trans-acting factors such as CRP, we also tested
reporters that contained only minimal core promoter
sequences lacking known binding sites [relAP1(242+2) and
relAP2(241+2); see Fig. 1a]. The minimal relAP1(242+2)
reporter remained sensitive to 6S RNA, similar to the
longer relAP1(2100+50) (compare Fig. 1d and Fig. 1b). The
minimal relAP2(241+2) reporter also remained sensitive to
6S RNA, although to a lesser extent than the reporter that
also contained the CRP binding site (relAP2(2100+50))
(compare Fig. 1e and Fig. 1c). In agreement with CRP
activation at this promoter, the overall level of expression of the minimal relAP2(241+2) was decreased relative to
relAP2(2100+50). hns has also been reported to activate
transcription from relAP2 (Nakagawa et al., 2006).
However, we observed a decrease in hns mRNA levels in
cells lacking 6S RNA compared with wild-type cells (A. T.
Cavanagh & K. M. Wassarman, unpublished data),
suggesting that a 6S RNA-dependent decrease in hns
cannot account for the observed 6S RNA-dependent
increase in relA expression.