RNA stem-loop enhanced expression of previously non-expressible genes.
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; DeltaG(0) = -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.
Paulus
Michael
M
Roche Diagnostics, Nonnenwald 2, D-82377 Penzberg, Germany.
Haslbeck
Martin
M
Watzele
Manfred
M
D001483:Base Sequence; D001665:Binding Sites; D005786:Gene Expression Regulation; D005818:Genetic Engineering; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D009690:Nucleic Acid Conformation; D010442:Peptide Chain Initiation, Translational; D014176:Protein Biosynthesis; D011506:Proteins / Q000032:analysis / Q000235:genetics; D020871:RNA Stability; D012333:RNA, Messenger / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D038621:Regulatory Sequences, Ribonucleic Acid / Q000235:genetics*; D016914:Ribonuclease H / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism; D014158:Transcription, Genetic / Q000235:genetics
D011506:Proteins; D012333:RNA, Messenger; D038621:Regulatory Sequences, Ribonucleic Acid; D016914:Ribonuclease H