Efficient expression of a protein coding gene under the control of an RNA polymerase I promoter

• Published in: Nucleic acids research Vol. 21; no. 15; pp. 3451 - 3457
• Main Authors: Palmer, Theo D., Miller, A.Dusty, Reeder, Ronald H., McStay, Brian
• Format: Journal Article
• Language: English
• Published: Oxford Oxford University Press 25.07.1993
• Subjects: Animals; Base Sequence; Biological and medical sciences; Biotechnology; Cell Line; Cloning, Molecular; Deoxyribonuclease EcoRI; DNA, Ribosomal - genetics; Drug Resistance - genetics; Fundamental and applied biological sciences. Psychology; Gene Expression; Genetic engineering; Genetic technics; Humans; Kanamycin Kinase; Methods. Procedures. Technologies; Molecular Sequence Data; Mutagenesis, Site-Directed; Neomycin; Phosphotransferases - genetics; Plasmids; Poly A - genetics; Promoter Regions, Genetic; Rats; RNA Polymerase I - metabolism; Simian virus 40; Simian virus 40 - genetics; Transcription, Genetic; Transfection; Vectors (cloning, transfer, expression). Insertion sequences and transposons; Performance evaluation; 5' terminal-Sequence; RNA polymerase 1; Enzyme; Transcription promoter; Optimization method; Genetical translation; Vector; IS element
• ISSN: 0305-1048; 1362-4962
• DOI: 10.1093/nar/21.15.3451

In mammalian cells, RNA polymerase I transcripts are uncapped and retain a polyphosphate 5′ terminus. It is probably for this reason that they are poorly translated as messenger RNA. We show in this report that insertion of an internal Ribosome Entry Site (IRES) into the 5′ leader of an RNA polymerase I transcript overcomes the block to translation, presumably by substituting for the 5′ trimethyl G cap. Addition of an SV40 polyA addition signal also enhances protein production from the RNA polymerase I transcript. RNA Polymerase I driven expression vectors containing both elements produce protein at levels comparable to that produced from RNA polymerase II driven expression vectors which utilize a retroviral LTR. RNA Polymerase I driven expression vectors may have a variety of uses both for basic research and for practical expression of recombinant proteins.