The influence of adenine-rich motifs in the 3' portion of the ribosome binding site on human IFN-gamma gene expression in Escherichia coli.
The ribosome binding site (RBS) of prokaryotic mRNA is divided into 5' and 3' portions by the translation initiation codon. Although it is well known that the presence of an appropriate RBS containing only the 5' portion is sufficient to direct the initiation of protein synthesis, the 3' portion appears to play a significant role in modulating the initiation process as well. Here we examine the influence of adenine-rich motifs frequently found in the 3' portion of highly expressed prokaryotic mRNAs. Two synthetic DNA fragments, GAGAAAAAAATC (corresponding to the first 12 nucleotides following the initiation codon of the chloramphenicol acetyltransferase gene), and AAAAAAATTAA were used to modify the beginning of the coding region of the human immune interferon-gamma (IFN-gamma) gene. The level of the protein synthesis in Escherichia coli directed by plasmids containing these constructs was quantitated. We found that placing either adenine-rich motif in the 3' portion of the RBS strongly enhanced gene expression, probably through an effect on translation initiation. We have also compared the protein expression levels of these gene constructs containing different series of 5'-RBSs with varying precistronic lengths and Shine-Dalgarno sequence lengths. The results suggest a positive functional role for the 3' adenine-rich motif. A possible mechanism for these effects is discussed.
Chen
H
H
Department of Chemistry, University of New Brunswick Fredericton, Canada.
Pomeroy-Cloney
L
L
Bjerknes
M
M
Tam
J
J
Jay
E
E
D000225:Adenine; D001483:Base Sequence; D001665:Binding Sites; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics*; D003001:Cloning, Molecular / Q000379:methods; D003062:Codon / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005796:Genes; D006801:Humans; D007371:Interferon-gamma / Q000096:biosynthesis* / Q000235:genetics*; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D012322:RNA Precursors / Q000096:biosynthesis; D012333:RNA, Messenger / Q000096:biosynthesis; D015183:Restriction Mapping; D012270:Ribosomes / Q000378:metabolism*
D003062:Codon; D009838:Oligodeoxyribonucleotides; D012322:RNA Precursors; D012333:RNA, Messenger; D007371:Interferon-gamma; D015500:Chloramphenicol O-Acetyltransferase; D000225:Adenine