Published online 26 May 2004
Nucleic Acids Research, 2004, Vol. 32, No. 9 e78
RNA stem–loop enhanced expression of previously non-expressible genes
Roche Diagnostics, Nonnenwald 2, D-82377 Penzberg, Germany and 1 Institut für Organische Chemie und Biochemie, Fakultät für Chemie, Technische Universität München, D-85747 Garching, Germany
*To whom correspondence should be addressed. Tel: +49 8856 603121; Fax: +49 8856 607609; Email: Manfred.Watzele{at}Roche.com
Received December 10, 2003; Revised February 21, 2004;; Accepted May 4, 2004
The key step in bacterial translation is formation of the pre-initiation complex. This requires initial contacts between mRNA, fMet-tRNA and the 30S subunit of the ribosome, steps that limit the initiation of translation. Here we report a method for improving translational initiation, which allows expression of several previously non-expressible genes. This method has potential applications in heterologous protein synthesis and high-throughput expression systems. We introduced a synthetic RNA stem–loop (stem length, 7 bp; 
G0 = –9.9 kcal/mol) in front of various gene sequences. In each case, the stem–loop was inserted 15 nt downstream from the start codon. Insertion of the stem–loop allowed in vitro expression of five previously non-expressible genes and enhanced the expression of all other genes investigated. Analysis of the RNA structure proved that the stem–loop was formed in vitro, and demonstrated that stabilization of the ribosome binding site is due to stem–loop introduction. By theoretical RNA structure analysis we showed that the inserted RNA stem–loop suppresses long-range interactions between the translation initiation domain and gene-specific mRNA sequences. Thus the inserted RNA stem–loop supports the formation of a separate translational initiation domain, which is more accessible to ribosome binding.