The whole set of constitutive promoters recognized by RNA polymerase RpoD holoenzyme of Escherichia coli

Abstract

The promoter selectivity of Escherichia coli RNA polymerase is determined by the sigma subunit with promoter recognition activity. The model prokaryote Escherichia coli contains seven species of the sigma subunit, each recognizing a specific set of promoters. The major sigma subunit, sigma-70 encoded by rpoD, plays a major role in transcription of growth-related genes. Concomitant with the increase in detection of promoters functioning in vivo under various stressful conditions, the variation is expanding in the consensus sequence of RpoD promoters. In order to identify the canonical sequence of "constitutive promoters" that are recognized by the RNA polymerase holoenzyme containing RpoD sigma in the absence of supporting transcription factors, an in vitro mixed transcription assay was carried out using a whole set of variant promoters, each harboring one base replacement, within the model promoter with the conserved -35 and -10 sequences of RpoD promoters. The consensus sequences, TTGACA(-35) and TATAAT(-10), were identified to be ideal for the maximum level of open complex formation and the highest rate of promoter opening, respectively. For identification of the full range of constitutive promoters on the E. coli genome, a total of 2,701 RpoD holoenzyme-binding sites were identified by Genomic SELEX screening, and using the reconfirmed consensus promoter sequence, a total of maximum 669 constitutive promoters were identified, implying that the majority of hitherto identified promoters represents the TF-dependent "inducible promoters". One unique feature of the constitutive promoters is the high level of promoter sequence conservation, about 85% carrying five-out-of-six agreements with -35 or -10 consensus sequence. The list of constitutive promoters provides the community resource toward estimation of the inducible promoters that operate under various stressful conditions in nature.

Figure 1. Distribution and classification of the constitutive promoters.

Genomic SELEX search of RpoD holoenzyme-binding sequences was performed using the standard procedure . RpoD holoenzyme-bound DNA fragments were recovered by immunoprecipitation using anti-RpoC antibody. SELEX fragments were isolated from the immuno-precipitates and subjected to mapping on the E. coli genome by using tilling DNA microarray as described previously , . [A] Location of the constitutive promoters. A total of 2,701 RpoD holoenzyme-binding sites were identified (see Fig. 1), of which 1,075 (40%) are located within intergenic spacers. On the basis of transcription direction of flanking genes, the spacers were classified into three types: type-A between bidirectional transcription units; type-B upstream of one transcription unit but downstream of another transcription unit; and type-C, downstream of both transcription units. [B] Classification of the constitutive promoters. A total of 2,082 promoters have been identified and listed in the current versions of RegulonDB and EcoCyc databases, whereas the total number of constitutive promoters identified by Genomix SELEX screening ranges between minimum 492 and maximum 669, indicating that the majority of E. coli promoters listed in promoter database are TF-dependent inducible promoters.

Figure 2. RpoD holoenzyme-binding peaks within type-A spacers.

RpoD holoenzyme-binding peaks were identified within a total of 177 type-A spacers. Some representative patterns of RpoD holoenzyme-peaks are shown, which are located between leuL-leuO (a), csgD-csgB (b), nanC-fimB (c), lrhA-alaA (d), adhE-ychE (e), waaQ-waaA (f), mdoC-mdoG (g) and ulaG-ulaA (h). Distribution of promoter -35 (indicated by orange arrows) and -10 (indicated by green arrows) signals is shown in the panel under each SELEX pattern.

Figure 3. RpoD holoenzyme-binding peaks within type-B spacers.

RpoD holoenzyme-binding peaks were identified within a total of 315 type-B spacers. Some representative patterns of RpoD holoenzyme-peaks are shown, which are located, which include the constitutive promoters for cydA (a), yfcV (b), yobF (c), ompT (d), yjeJ (e), yhcN (f), yfjL (g) and phoH (h) operons. Distribution of promoter -35 and -10 is shown below each panel.

Figure 4. Determination of the consensus sequence of constitutive promoters using the in vitro mixed transcription system.

Mixtures of equal amounts of 195 bp-long template containing the ideal promoter of complete consensus sequence and 175 bp-long mutant template, each carrying one base substitution, were subjected to the in vitro mixed transcription , . After preincubation for 0.5, 1.0, 2.5, 5.0, 7.5, 10 and 15 min, a mixture of substrates and heparin was added and RNA synthesis was allowed for 10 min. The final level of RNA synthesis represents the level of RpoD holoenzyme binding (parameter I) while the rate of open complex formation (parameter II) was determined as a reciprocal of the time required to reach the plateau level. For each set of four promoters with mutations at the same position, the promoter activities are shown as the values relative to the promoter with the highest activity.

Figure 5. The composition of constitutive promoters.

[A] The known consensus sequences of RpoD-dependent promoter, TTGAAC (-35) and TATAAT (-10) separated by 17 plus/minus 2 bp, were searched for all type-A (177) and type-B (315) spacers (see Experimental Procedure for the analysis method). Most of the constitutive promoters carry high-levels of the consensus sequence as listed in Table 1. The composition of promoter -35 and -10 sequence was classified into 8 groups based on the conservation level of consensus sequences. About 89% of type-A promoters and 82% of type-B promoters (or 86% of A- plus B-type promoters) contain the sequence higher than 4/6 agreement with the consensus sequence at both -35 or -10 positions (A1), while only 39% of a total of 582 known promoters carries this level of consensus sequences (B1). [B] The whole set of constitutive promoter sequences were subjected to Logo analysis . The Logo patterns of -35 and -10 sequences are essentially the same among the constitutive promoters within type-A and type-B spacers. The Logo pattern of the whole set of constitutive promoters was compared with the Logo pattern generated using the total of 582 experimentally identified promoters , . The contribution of each base of the consensus -35 and -10 sequences is significantly different between the constitutive promoters and the set of known promoters.