Regulation of gene expression at the level of translation accounts for up to three orders of magnitude in its efficiency. We systematically compared the impact of several mRNA features on translation initiation at the first gene in an operon with those for the second gene. Experiments were done in a system with internal control based on dual cerulean and red (CER/RFP) fluorescent proteins. We demonstrated significant differences in the efficiency of Shine Dalgarno sequences acting at the leading gene and at the following genes in an operon. The majority of frequent intercistronic arrangements possess medium SD dependence, medium dependence on the preceding cistron translation and efficient stimulation by A/U-rich sequences. The second cistron starting immediately after preceding cistron stop codon displays unusually high dependence on the SD sequence.