Coordinated, Differential Expression of Two Genes through Directed mRNA Cleavage and Stabilization by Secondary Structures

Abstract

Metabolic engineering and multisubunit protein production necessitate the expression of multiple genes at coordinated levels. In bacteria, genes for multisubunit proteins or metabolic pathways are often expressed in operons under the control of a single promoter; expression of the genes is coordinated by varying transcript stability and the rate of translation initiation. We have developed a system to place multiple genes under the control of a single promoter and produce proteins encoded in that novel operon in different ratios over a range of inducer concentrations. RNase E sites identified in the Rhodobacter capsulatus puf operon and Escherichia coli pap operon were separately placed between the coding regions of two reporter genes, and novel secondary structures were engineered into the 5 and 3 ends of the coding regions. The introduced RNase E site directed cleavage between the coding regions to produce two secondary transcripts, each containing a single coding region. The secondary transcripts were protected from exonuclease cleavage by engineered 3 secondary structures, and one of the secondary transcripts was protected from RNase E cleavage by secondary structures at the 5 end. The relative expression levels of two reporter genes could be varied up to fourfold, depending on inducer concentration, by controlling RNase cleavage of the primary and secondary transcripts. Coupled with the ability to vary translation initiation by changing the ribosome binding site, this technology should allow one to create new operons and coordinate, yet separately control, the expression levels of genes expressed in that operon.

Production and secretion of fatty acids in genetically engineered cyanobacteria

“…The nucleic acid sequences of non-6803 genes were redesigned by codon optimization on the basis of the codon frequencies of highly expressed 6803 genes (Table S4). Also, the stem-loop hairpins in the predicted mRNA secondary structure were removed to smooth the transcription and to stabilize mRNA by prolonging its half-life (36).…”

Fatty acid production in genetically modified cyanobacteria

“…The nucleic acid sequences of foreign genes were redesigned by codon optimization on the basis of the codon frequencies of highly expressed Synechocystis genes (Table S4). Also stem-loop hairpins in the predicted mRNA secondary structure were removed to stabilize mRNA by prolonging its half-life (43).…”

Continuous Control of the Flow in Biochemical Pathways through 5′ Untranslated Region Sequence Modifications in mRNA Expressed from the Broad-Host-Range Promoter Pm

“…For example, when the goal is to optimize the expression level of a desired protein by engineering the relevant metabolic pathway, it might be necessary to change the expression of multiple enzymes simultaneously and to different levels (30,38). Also, reducing the formation of particular by-products can increase the flux of the desired product (29).…”