Modification of mRNA secondary structure and alteration of the expression of human interferon alpha 1 in Escherichia coli.
A plasmid (pNL015) was constructed to contain a human interferon alpha 1 (IFN-alpha 1) gene under the transcriptional control of the Escherichia coli lipoprotein promoter. The E. coli cells harboring this plasmid produce 2.8 x 10(4) units/ml of IFN. Secondary structure analysis of the transcripts produced by pNL015 showed that the coding region could base pair with the Shine-Dalgarno (SD) region with a delta G = -3.9kcal/mol. A new plasmid pNL008 was constructed by modifying pNL015 with an 11-bp deletion and a 2-bp insertion in the coding region, so that the SD region is not involved in the secondary structure. E. coli cells harboring pNL008 produce ten times more IFN activity than cells harboring pNL015. A series of experiments were carried out to show that the specific activities of IFN, differential rates of IFN transcription, protein degradation or mRNA degradation could not account for the difference observed in expression. A rigorous test on this model of translational inhibition was conducted by the construction of pNL017 with a single bp substitution which did not change the amino acid sequence of the IFN (synonymous codon substitution) but which increased the calculated energy of interaction with the SD sequence to delta G = -10.8 kcal/mol. The E. coli cells harboring pNL017 produced no detectable IFN activity.
Lee
N
N
Interferon Sciences, Inc., New Brunswick, NJ 08901.
Zhang
S Q
SQ
Cozzitorto
J
J
Yang
J S
JS
Testa
D
D
D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics*; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D006801:Humans; D007370:Interferon Type I / Q000096:biosynthesis / Q000235:genetics*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010957:Plasmids; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics*
D007370:Interferon Type I; D012333:RNA, Messenger