Shen, W.-Y. | bioRxiv Science

bioRxiv2025

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 PluxI, 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.

systems biology↗

bioRxiv2025

Unravel the start element and promoter architecture across the domain Bacteria

Core promoters comprise multiple elements whose interaction with RNA polymerase initiates transcription. Despite decades of research, substantial sequence and length variation of promoter elements has hindered efforts to elucidate their function and the evolutionary diversity of transcriptional regulation. Combining massively parallel assays, biophysical modeling, and functional validation, we systematically dissected the promoter architecture upstream of experimentally determined transcription start sites in 49 phylogenetically diverse bacterial genomes (GC content: 27.8-72.1%). We identified a conserved 3-bp promoter element, termed start, that dictates transcription start site selection and enhances transcription. We uncovered a four-region organization within the variable spacer element, whose sequence composition modulates transcription by up to 600-fold. We showed that the discriminator element is conserved in Terrabacteria but diverse in Gracilicutes, the two major bacterial clades. High discriminator sequence diversity in Gracilicutes likely reflects diversifying evolution, enabling promoter-encoded regulation to orchestrate global gene expression in response to growth rate changes. Together, our findings reveal broad conservation of bacterial promoter organization while highlighting regulatory divergence of promoter elements and RNA polymerase between Terrabacteria and Gracilicutes. Sequence and functional similarities between bacterial promoter elements and their archaeal and eukaryotic counterparts further suggest a shared evolutionary origin of promoter architecture.

microbiology↗