Transcription Activation at Promoters Carrying Tandem DNA Sites for the Escherichia coli Cyclic AMP Receptor Protein: Organisation of the RNA Polymerase a Subunits Tamara A. Belyaeva, Virgil A. Rhodius, Christine L. Webster and Stephen J. W. Busby* School of Biochemistry University of Birmingham Birmingham B15 2TT, UK We have constructed a family of promoters carrying tandem DNA sites for the Escherichia coli cyclic AMP receptor protein (CRP), with one of the sites centred between base-pairs 41 and 42 upstream from the transcrip- tion start site, and the second site located further upstream. In vivo activity measurements show that the activity of these promoters is com- pletely dependent on CRP and that, depending on the precise location, CRP bound at the upstream site increases transcription activation. Hydroxyl radical footprinting was exploited to investigate the binding of CRP and RNA polymerase holoenzyme (RNAP) to these promoters. The study shows that the C-terminal domains of the RNAP a subunits bind adjacent to the upstream CRP and that their precise positioning depends on the location of upstream-bound CRP. The C-terminal domains of the RNAP a subunits interact with both the upstream and downstream- bound CRP via activating region 1 of CRP. # 1998 Academic Press Limited Keywords: cyclic AMP receptor protein; Escherichia coli; transcription activation; activating regions; RNA polymerase a subunit *Corresponding author Introduction Hundreds of bacterial promoters are subject to regulation by transcription activators. In some cases, promoter activity is dependent solely on a single activator whilst, in other cases, promoter activity is co-dependent on two or more activators that function together synergistically. Over the past decade, the mechanisms by which promoters are activated, and by which promoter function can be coupled to multiple activators, have been subjected to intense scrutiny. Most bacterial transcription activators function by making direct interactions with holo RNA polymerase (RNAP) at target pro- moters, with the activator±RNAP interaction sti- mulating transcription initiation. Escherichia coli RNAP has been found to contain many discrete contact sites for activators, with different activators interacting with different contact sites. This suggests a simple mechanism for regulating the activity of a promoter by two activators: the two activators make independent interactions with two different contact sites in RNAP and, thus, by mak- ing independent interactions with RNAP, two tran- scription activators can function synergistically at a target promoter (reviewed by Hochschild & Joung, 1997). For example, some promoters are co-regu- lated by one transcription activator that interacts with a target in the RNAP s subunit and a second activator that interacts with a target in the C-term- inal domains of the two RNAP a subunits (aCTD) (Joung et al., 1994; Scott et al., 1995). At these pro- moters, the binding site for the activator that inter- acts with s overlaps the 35 element, whilst the binding site for the activator that interacts with aCTD is located further upstream. In a recent study, Blatter et al. (1994) showed that aCTD is connected to the N-terminal domain of the RNA polymerase a subunit (aNTD) by a ¯exible linker, and suggested that this linker allows great vari- ation in the location of binding sites for activators that interact with aCTD. Thus, at promoters depen- dent on two activators, it is also likely that there is variation in the possible locations of the binding Abbreviations used: CRP, cyclic AMP receptor protein; RNAP, holo form of Escherichia coli DNA- dependent RNA polymerase; aCTD, C-terminal domain of RNA polymerase a subunit; aNTD, N-terminal domain of RNA polymerase a subunit. J. Mol. Biol. (1998) 277, 789±804 0022±2836/98/140789±16 $25.00/0/mb981666 # 1998 Academic Press Limited