The effects of endoribonuclease sites, secondary structures in mRNA, and gene placement on protein production and mRNA stability and steady-state levels were tested in a dual-gene operon containing the genes encoding β-galactosidase (lacZ) from Escherichia coli and green fluorescent protein (gfp) from Aequorea victoria. Two previously identified RNase E sites were placed separately between the coding regions to direct cleavage in this area and produce two secondary transcripts, each containing a single-gene coding region. Novel secondary structures were engineered into the 3′ and 5′ ends of each of the coding regions to protect the transcript from inactivation by endoribonucleases (5′ hairpins) and degradation by exoribonucleases (3′ hairpins). In addition, the effects of relative gene placement were examined by switching the locations of the two coding regions. Depending on the particular secondary structures and RNase E sites placed between the genes the relative steady-state transcript and protein levels encoded by the two reporter genes could be changed up to 2.5-fold and 4-fold, respectively. By changing gene location and incorporating secondary structures and RNase E sites the relative steady-state transcript and protein levels encoded by the two reporter genes could be changed up to 100-fold and 750-fold, respectively. © 2002 Wiley Periodicals, Inc. Biotechnol Bioeng 80: 762–776, 2002.