Coordinated, differential expression of two genes through directed mRNA cleavage and stabilization by secondary structures

Metabolic engineering and multisubunit protein production necessitate the e xpression of multiple genes at coordinated levels. In bacteria, genes for m ultisubunit proteins or metabolic pathways are often expressed in operons u nder the control of a single promoter; expression of the genes is coordinat ed by varying transcript stability and the rate of translation initiation, We have developed a system to place multiple genes under the control of a s ingle promoter and produce proteins encoded in that novel operon in differe nt ratios over a range of inducer concentrations. RNase E sites identified in the Rhodobacter capsulatus puf operon and Escherichia coli pap operon we re separately placed between the coding regions of two reporter genes, and novel secondary structures were engineered into the 5' and 3' ends of the c oding regions. The introduced RNase E site directed cleavage between the co ding regions to produce two secondary transcripts, each containing a single coding region. The secondary transcripts were protected from exonuclease c leavage by engineered 3' secondary structures, and one of the secondary tra nscripts was protected from RNase E cleavage by secondary structures at the 5' end. The relative expression levels of two reporter genes could be vari ed up to fourfold, depending on inducer concentration, by controlling RNase cleavage of the primary and secondary transcripts. Coupled with the abilit y to vary translation initiation by changing the ribosome binding site, thi s technology should allow one to create new operons and coordinate, yet sep arately control, the expression levels of genes expressed in that operon.