(18,21,22) These σ factors are differentiated by both the protein domains present within the protein itself and by the DNA sequences they bind to. (19,20) Within the σ 70 protein family, there are additional σ factors that respond to cellular stresses such as σ 38/S, σ 28/F, σ 32/H, σ 24/E, and σ 19/FecI. (17) Promoter elements interacting with the housekeeping σ 70, also known as σ D, are the most well-characterized (18) and have been demonstrated to bind specifically with their target promoters through DNA–protein interactions such as hydrogen bonding, van der Waals forces, and stacked cation−π bonds. RNAP holoenzyme interactions can be enhanced or replaced through accessory sequences, such as the extended −10 TGn motif and upstream (UP) elements, which interact with the bacterial σ factor and α subunits, respectively.
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While the −35 and −10 elements are the canonical drivers of transcription, promoter DNA–protein interactions are not limited to the two core elements.
Transcription is initiated when a σ factor binds to dsDNA at the −35 element, promoting DNA isomerization to ssDNA at the −10 element, which drives transcription events. The core elements bind to the primary specification component within the RNA polymerase (RNAP) holoenzyme, the σ 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.īacterial promoter sequences are typically made of two core sequence elements, the −35 and −10 elements. CORPSE exploits the DNA-σ factor structural relationship to disrupt σ 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 σ factors and eliminate downstream transcriptional activity. The promoter activity is dependent on the structural interaction of core bases with a σ 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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