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(19,20) Within the Пѓ 70 protein family, there are additional Пѓ factors that respond to cellular stresses such as Пѓ 38/S, Пѓ 28/F, Пѓ 32/H, Пѓ 24/E, and Пѓ 19/FecI. (17) Promoter elements interacting with the housekeeping Пѓ 70, also known as Пѓ D, are the most well-characterized (18) and have been demonstrated to bind specifically with their target promoters through DNAвЂ“protein interactions such as hydrogen bonding, van der Waals forces, and stacked cationв€’ПЂ bonds. RNAP holoenzyme interactions can be enhanced or replaced through accessory sequences, such as the extended в€’10 TGn motif and upstream (UP) elements, which interact with the bacterial Пѓ factor and О± subunits, respectively.

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While the в€’35 and в€’10 elements are the canonical drivers of transcription, promoter DNAвЂ“protein interactions are not limited to the two core elements. Transcription is initiated when a Пѓ factor binds to dsDNA at the в€’35 element, promoting DNA isomerization to ssDNA at the в€’10 element, which drives transcription events. The core elements bind to the primary specification component within the RNA polymerase (RNAP) holoenzyme, the Пѓ factor. Additionally, we present an inverted CORPSE system, iCORPSE, which can create highly active promoters within a gene sequence while not perturbing the function of the modified gene.Д«acterial promoter sequences are typically made of two core sequence elements, the в€’35 and в€’10 elements.

CORPSE exploits the DNA-Пѓ factor structural relationship to disrupt Пѓ 70 promoters embedded within gene coding sequences with a minimum of synonymous codon changes. In this paper, we present codon-restrained promoter silencing (CORPSE), a system for removing intragenic promoters. Rational engineering can be used to alter key promoter element nucleotides interacting with Пѓ factors and eliminate downstream transcriptional activity. The promoter activity is dependent on the structural interaction of core bases with a Пѓ factor. These regulatory elements include cryptic and intragenic promoters, which may constitute up to a third of the predicted Escherichia coli promoters. Future applications of synthetic biology will require refactored genetic sequences devoid of internal regulatory elements within coding sequences.

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