Recognition Of Cis-regulatory Elements Research Articles

Engineering Synthetic cis-Regulatory Elements for Simultaneous Recognition of Three Transcriptional Factors in Bacteria.

Recognition of cis-regulatory elements by transcription factors (TF) at target promoters is crucial to gene regulation in bacteria. In this process, binding of TFs to their cognate sequences depends on a set of physical interactions between these proteins and specific nucleotides in the operator region. Previously, we showed that in silico optimization algorithms are able to generate short sequences that are recognized by two different TFs of Escherichia coli, namely, CRP and IHF, thus generating an AND logic gate. Here, we expanded this approach in order to engineer DNA sequences that can be simultaneously recognized by three unrelated TFs (CRP, IHF, and Fis). Using in silico optimization and experimental validation strategies, we were able to obtain a candidate promoter (Plac-CFI1) regulated by only two TFs with an AND logic, thus demonstrating a limitation in the design. Subsequently, we modified the algorithm to allow the optimization of extended sequences, and were able to design two synthetic promoters (PCFI20-1 and PCFI22-5) that were functional in vivo. Expression assays in E. coli mutant strains for each TF revealed that while CRP positively regulates the promoter activities, IHF and Fis are strong repressors of both the promoter variants. Taken together, our results demonstrate the potential of in silico strategies in bacterial synthetic promoter engineering. Furthermore, the study also shows how small modifications in cis-regulatory elements can drastically affect the final logic of the resulting promoter.

Identification of a cis-acting factor modulating the transcription of FUM1, a key fumonisin-biosynthetic gene in the fungal maize pathogen Fusarium verticillioides

Fumonisins, toxic secondary metabolites produced by some Fusarium spp. and Aspergillus niger, have strong agro-economic and health impacts. The genes needed for their biosynthesis, named FUM, are clustered and co-expressed in fumonisin producers.In eukaryotes, coordination of transcription can be attained through shared transcription factors, whose specificity relies on the recognition of cis-regulatory elements on target promoters. A bioinformatic analysis on FUM promoters in the maize pathogens Fusarium verticillioides and Aspergillus niger identified a degenerated, over-represented motif potentially involved in the cis-regulation of FUM genes, and of fumonisin biosynthesis. The same motif was not found in various FUM homologues of fungi that do not produce fumonisins. Comparison of the transcriptional strength of the intact FUM1 promoter with a synthetic version, where the motif had been mutated, was carried out in vivo and in planta for F. verticillioides. The results showed that the motif is important for efficient transcription of the FUM1 gene.