Figure 4. Determination of transcription units (TUs) and use of alternative TSSs.

(a) Modular units (MU, blue horizontal bars) are assembled in a condition-dependent manner, resulting in different TUs (green arrow). Under log phase growth conditions, modular units FWD-1 (containing thrA) and FWD-2 (containing thrBC) are transcribed together forming contiguous TUs (TU-1,2,3, based on transcription start site (TSS) information). However, stationary growth phase triggers transcription of modules FWD-1 and FWD-2 separately, defining an additional TU (TU-4). Module FWD-3, resulting in TU-5, is used similar under log and stationary phase. Dotted lines in the transcription profiles indicate change points of transcription. The change point under stationary phase (star) led to the determination of one additional TU (TU-4). (b) Regulatory elements responsible for differential usage of modular units were measured by the elucidation of σ⁷⁰ and σ^S holoenzyme (Eσ⁷⁰ and Eσ^S) occupancy within the promoter regions (i, ii) and control region (iii) in log and stationary phase, respectively (see Methods). Significant occupation preferences of σ⁷⁰ and σ^S holoenzymes confirmed the convoluted TU architecture. (c) Number of transcription start sites per promoter region. We observed that ∼35% of promoter regions contain multiple TSSs, indicating the presence of alternative TSSs for large portions of the E. coli TU architecture. (d) The stpA gene and the livKHMGF operon have multiple experimentally verified TSSs. We detected the dominant TSS (2,796,558) for the stpA promoter, which is highly activated by the transcription factor Lrp^15,22. Therefore, the other two experimentally confirmed TSSs (2,796,578 and 2,796,600) are likely to be utilized less under this growth condition. The transcription factor Lrp also represses one TSS (3,595,778) of the livK promoter. The other previously confirmed TSS (3,595,753) was observed to be the dominant TSS. (e) The transcription factors Fnr and ArcA repress transcription of the nuo operon under anaerobic conditions⁹. Since the transcription factors are inactivated under aerobic conditions, we confirmed the existence of previously confirmed TSS (2,403,219), which is specifically released from the repression.