PMID:15163763
| Citation |
Paulus, M, Haslbeck, M and Watzele, M (2004) RNA stem-loop enhanced expression of previously non-expressible genes. Nucleic Acids Res. 32:e78 |
|---|---|
| Abstract |
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. |
| Links |
PubMed PMC419630 Online version:10.1093/nar/gnh076 |
| Keywords |
Base Sequence; Binding Sites; Gene Expression Regulation; Genetic Engineering; Molecular Sequence Data; Mutation/genetics; Nucleic Acid Conformation; Peptide Chain Initiation, Translational; Protein Biosynthesis; Proteins/analysis; Proteins/genetics; RNA Stability; RNA, Messenger/chemistry; RNA, Messenger/genetics; RNA, Messenger/metabolism; Regulatory Sequences, Ribonucleic Acid/genetics; Ribonuclease H/metabolism; Ribosomes/metabolism; Transcription, Genetic/genetics |
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