The effects of DNA sequence on interferon mRNA biosynthesis and stability in E. coli

• Main Author: Johnston, Julie Sharon
• Published: Royal Holloway, University of London 1987
• Subjects: 572; Biochemistry
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.704417

The aim of this investigation was determine the effect of DNA sequences on interferon mRNA biosynthesis and stability in E.coli. Interferon al and a2 DNA sequences are 80% homologous. Despite this large degree of similarity different levels of protein and different transcription products were obtained when the two genes were cloned into identical vectors. Increasing the plasmid copy number and introducing a transcription terminator immediately downstream from the interferon sequence increased al production. Deleting the sequence upstream from the -35 region of the promoter or reducing the length of the 3' non-coding region had little effect on a2 production. Highest levels of a2 interferon were obtained by altering the ribosome binding site. This necessitated decreasing the plasmid copy number to achieve maximum plasmid stability. In contrast, all al containing plasmids were stability maintained in the bacteria. Analysis of the transcription products of all the interferon producing clones revealed that multiple mRNA species were generated by all the constructs except those which contained the termination signal. Removal of the sequence 5' to the -35 region of the promotor resulted in reduced transcription, while shortening the 3' non-coding region lowered termination efficiency within the remaining sequence facilitating the production of polycistronic messages. Stability studies were undertaken to determine the half-lives of the mRNA species. Most decayed with a half-life of 1-1.5 minutes. However, more stable species were produced by the constructs containing the termination signal. Comparison of the mRNA species produced by Similar al and a2 producing strains revealed that while all the al and a2 mRNA were of sufficient length for translation to yield mature interferon three a2 mRNA species appeared too short. These prematurely terminating mRNA were further characterized. The smallest terminated at a rho-dependant termination site while the other two species terminated at rho-independant sites. Termination at the rho-dependant site could be eliminated if the rare arginine codons AGG present in the early coding sequence were replaced by the more commonly used arginine codons CGT. Transcription profiles and mRNA half-lives were determined in different media at different growth rates to see whether production of the prematurely terminating species was affected. While all species were produced under all conditions the distribution of mRNA species altered with both media and growth phase.