RNA primary sequence or secondary structure in the translational initiation region controls expression of two variant interferon-beta genes in Escherichia coli.
Efficient expression in Escherichia coli (E. coli) of the human interferon-beta gene (IFN-beta) gene and of a chemically synthesized IFN-beta gene variant (506 base pairs; synIFN-beta) adapted to the E. coli codon usage, both fused to the E. coli atpE ribosome-binding site, is controlled either by primary sequence or by mRNA secondary-structure in the translational initiation region. High level expression of the natural human atpE/IFN-beta gene fusion is governed by the nucleotide composition preceding the initiator codon AUG. A single U----C exchange in the -2 or -1 position preceding the initiator codon AUG reduces the translational efficiency from 18% of total cellular protein to only 8% or 4%, respectively, while both U----C substitutions reduce IFN-beta expression below 1%. These sequence alterations interfere with efficient ribosome binding as revealed by toeprinting. They provide further evidence for the influence of the anticodon-flanking regions of tRNA(fMet) upon the initiation rate of translation. In contrast, translation of the synthetic variant atpE/synIFN-beta gene fusion is controlled by a moderately stable stem-loop structure (delta G = -4 kcal/mol; 37 degrees C) located within the coding region and overlapping the 30 S ribosomal subunit attachment site. That the stability of the hairpin interferes with the initiation of translation is inferred from site-directed mutagenesis and toeprint analyses. mRNA half-life in these variants is positively correlated with the rate of translation and involves two major endonucleolytic cleavage site 5'-upstream of the Shine-Dalgarno region.
Gross
G
G
Gesellschaft für Biotechnologische Forschung, Abteilung Genetik, Braunschweig, Federal Republic of Germany.
Mielke
C
C
Hollatz
I
I
Blöcker
H
H
Frank
R
R
D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005813:Genes, Synthetic; D014644:Genetic Variation; D006801:Humans; D007370:Interferon Type I / Q000235:genetics*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010442:Peptide Chain Initiation, Translational; D012333:RNA, Messenger / Q000235:genetics*; D012689:Sequence Homology, Nucleic Acid
D007370:Interferon Type I; D012333:RNA, Messenger