Study of in vitro transcriptional binding effects and noise using constitutive promoters combined with UP element sequences in Escherichia coli

Abstract

Abstract Background UP elements (upstream element) are DNA sequences upstream of a promoter that interact with the α-subunit of RNA polymerase (RNAP) and can affect transcription by altering the binding RNAP to DNA. However, details of UP element and binding affinity effects on transcriptional strength are unclear. Results Here, we investigated the effects of UP element sequences on gene transcription, binding affinity, and gene expression noise. Addition of UP elements resulted in increased gene expression (maximum 95.7-fold increase) and reduced gene expression noise (8.51-fold reduction). Half UP element sequences at the proximal subsite has little effect on transcriptional strength despite increasing binding affinity by 2.28-fold. In vitro binding assays were used to determine dissociation constants (Kd) and in the in vitro system, the full range of gene expression occurs in a small range of dissociation constants (25 nM < Kd < 45 nM) indicating that transcriptional strength is highly sensitive to small changes in binding affinity. Conclusions These results demonstrate the utility of UP elements and provide mechanistic insight into the functional relationship between binding affinity and transcription. Given the centrality of gene expression via transcription to biology, additional insight into transcriptional mechanisms can foster both fundamental and applied research. In particular, knowledge of the DNA sequence-specific effects on expression strength can aid in promoter engineering for different organisms and for metabolic engineering to balance pathway fluxes.

Keywords

• Upstream element sequence
• RNAP α subunits
• Gene expression noise
• Promoter strength
• RNA polymerase
• Binding association constant