Bacterial expression of rat liver succinyl-CoA synthetase alpha-subunit. Factors that contribute to blocked translation of transcripts encoding a mitochondrial signal sequence.
This study comprises a detailed evaluation of factors that are necessary to achieve high levels of expression of eukaryotic proteins in bacterial systems. We attempted to express a rat liver cDNA clone encoding the precursor to the alpha-subunit of succinyl-CoA synthetase in an Escherichia coli expression system, without success. Removal of the region encoding the mitochondrial signal peptide (115 nucleotides) allowed efficient expression of the mature protein. This nucleotide sequence was shown to block expression at the level of translation. Two regions within this fragment were able to block the expression of other genes such as E. coli lacZ. Inhibition of expression was due to the close proximity of these inhibitory sequences with the translation initiation region (TIR). Insertion of a spacer between the inhibitory sequence and the TIR relieved the block in translation. Analysis of the 115-nucleotide fragment identified sequences capable of extensive base-pairing with the Shine-Dalgarno and surrounding sequences. Such secondary structures are capable of blocking the formation of competent translation initiation complexes.
Ryan
D G
DG
Department of Biochemistry, University of Alberta, Edmonton, Canada.
Bridger
W A
WA
D000818:Animals; D001483:Base Sequence; D002151:Calorimetry; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D008099:Liver / Q000201:enzymology*; D046911:Macromolecular Substances; D008930:Mitochondria, Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D010957:Plasmids; D014176:Protein Biosynthesis; D021382:Protein Sorting Signals / Q000235:genetics*; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics; D051381:Rats; D015183:Restriction Mapping; D013389:Succinate-CoA Ligases / Q000235:genetics*; D014158:Transcription, Genetic
D046911:Macromolecular Substances; D021382:Protein Sorting Signals; D012333:RNA, Messenger; D013389:Succinate-CoA Ligases