Escherichia coli Promoters with UP Elements of Different Strengths: Modular Structure of Bacterial Promoters

Promoter sequences involved in recognition by Escherichia coli RNA polymerase (RNAP) were identified from comparisons of a large number of known promoters and from mutational analyses (28, 29, 38, 62). These sequences, the −10 and −35 hexamers (5′ TATAAT 3′ and 5′TTGACA 3′, respectively), are recognized by the ς70 subunit of RNAP (11). The strength of a promoter correlates generally with its degree of identity to these sequences and with the length of the spacer between them (the homology score [42]), although exceptions to this rule have been described (7, 26). It was proposed more than 10 years ago that optimal transcription activity could be achieved by different combinations of promoter elements, including not only the −10 and −35 hexamers, but also upstream and downstream regions (7). In accord with this suggestion, RNAP protects regions both upstream and downstream of the −10 and −35 hexamers in footprints (8, 45, 47, 56), and A+T-rich sequences upstream of the −35 hexamer in several E. coli or Bacillus subtilis promoters were found to increase transcription in vitro in the absence of accessory proteins (3, 19, 31, 37, 39, 50, 54). Phased A-tracts inserted upstream of the −35 region in various promoter constructs were also shown to increase transcription (6, 12, 24). The A+T-rich region upstream of −40 in the rRNA promoter rrnB P1, the UP element, increases transcription 30- to 70-fold by binding the RNAP α subunit (13, 50, 53). A consensus UP element sequence was determined by using in vitro selection for upstream sequences that promote rapid RNAP binding to the rrnB P1 promoter, followed by in vivo screening for high promoter activity. The consensus UP element consists of alternating A- and T-tracts (13). UP elements matching the consensus increased promoter activity as much as 326-fold, about 5-fold more than the wild-type rrnB P1 UP element. UP elements were also identified in other promoters, for example, the flagellin (hag) promoter of B. subtilis (18), the PL2 promoter of phage lambda (25), and the Pe promoter of phage Mu (61), although the effects of these elements were not as large as that of rrnB P1. UP elements also function in promoters recognized by RNAP holoenzymes with alternate ς factors (18). UP elements are not as highly conserved as the −10 and −35 elements and were not described in studies comparing the large sets of E. coli promoters used to define the consensus hexamers (28, 29, 38). However, A+T-rich sequences were identified as a prominent feature of a subset of E. coli promoters (the −44 motif [23]), and a recent E. coli promoter analysis (48) identified two A+T-rich regions at upstream positions corresponding to those crucial for UP element function (14). A+T-rich upstream sequences were also identified in compilations of B. subtilis and Clostridium promoters (27, 30). We have proposed that UP elements may be a recognition feature in many bacterial promoters (13, 53), but in most promoters, the role of upstream sequences has not been evaluated experimentally. Therefore, in this paper, we have examined the role of upstream sequences from six promoters (rrnB P2, rrnD P1, RNA II, merT, lac, and λ pR). We find that several of the sequences function as UP elements and that their effects on promoter activity differ, correlating generally with the degree of similarity to the UP element consensus sequence. These results support the model that bacterial promoters consist of at least three modules, not just −10 and −35 elements. We also show that upstream protection in footprints is not a reliable indicator of UP element function.