The introduction of an inverted repeat to the 5′ untranslated leader sequence of a transgene strongly inhibits gene expression

Abstract

Stable secondary structures in naturally occurring 5' untranslated regions have been shown to down-regulate both transcription and translation. We introduced a synthetic GC-rich inverted repeat to the leader sequence of a transgene to determine its influence on gene expression. The addition of a 54-bp inverted repeat led to a more than 90% reduction in transient gene expression, while the addition of an inverted repeat of 42-bp reduced gene expression by 88%. Complete removal of the inverted repeat abolished this inhibiting effect. This dramatic decrease in transgene expression is probably due to the formation of stable stem-loop structures in the 5' untranslated region of the reporter gene. The secondary structure energy of the putative stem-loop structures in the 54-bp and 42-bp repeats are -64.6 and -40.3 kcal mol-1, respectively. In comparison, the most stable stem-loop structure in the control construct's leader has a free energy of -15.4 kcal mol-1. This has important implications for the design of expression vectors where the recombination of multiple cloning sites and other 5' leader sequences can lead to the introduction of inverted repeats that has the potential of forming stable stem-loop structures and resulting in a significant decrease in gene expression.