Core elements play distinct roles in promoter birth and transcriptional regulation

Gene expression shapes phenotypes and evolution. However, studies of gene regulation focus on transcription factors, overlooking core promoters. To investigate how promoters emerge and regulate transcription, we determined the sequence–function landscapes of core elements, −35 and −10, in constitutive and transcription factor-regulated promoters in Escherichia coli . Characterization of in vivo transcriptional landscapes and in vitro RNA polymerase–promoter interactions showed the −10 element as essential for promoter evolution from random sequences. In contrast, the −35 element, though broadly conserved, is dispensable for promoter birth. Instead, it exerts greater impact on gene regulation via coordinated interactions with transcription activators and RNA polymerase. We further showed that evolution fine-tunes the −35 and −10 sequences of transcription factor-regulated promoters to achieve near-maximal fold changes by lowering basal while elevating induced expression. A notable exception is P luxI , whose leaky expression provides a crucial baseline for initiating quorum sensing. These findings elucidate promoter design principles and underscore the interdependence and coevolution of core elements, RNA polymerase, and transcription factors.