Abstract
Many proteins with unusual structural properties are comprised of multiple repeating amino acid sequences, and are often fractious to expression in recombinant systems. To facilitate recombinant production of such proteins for structural and engineering studies, the author has developed a method for producing messenger RNAs on circular RNA templates. This circularization process is derived from a rearranged group I intron, from which circular RNA is produced through the splicing activity of autocatalytic group I RNA elements (Fig. 1; 1,2). Because the only cofactors required for splicing of the group I intron are magnesium and guanosine, the process can take place in a variety of organisms, making it amenable to a wide variety of protein expression systems (1–4).
Design features of plasmids containing rearranged group I intron elements for circular GFP mRNA expression of monomeric GFP or polyGFP in (B) E. coli or (C) rabbit reticulocyte lysates. Transcription and splicing results in circularization of the bracketed sequence, between the 3′ (3′ss) and 5′ (5′ss) splice sites shown in each figure. (A) Relevant region of circular GFP mRNA plasmid containing the GFP ORF. Site of T7 RNA polymerase promoter sequence used for in vitro and in vivo (E. coli) RNA production is indicated. The 3′ and 5′ group I intron sequences are shown. AUG to UUA is the mutation introduced by the GFP-AUG oligonucleotide, to remove the initiating GFP-AUG (see Subheading 3.1. ) UAA to UAU is the mutation introduced by the GFP-stop oligonucleotide to remove the GFP-stop codon, thus allowing creation of infinite circular ORFs (see Subheading 3.1. ). (B) Circular GFP mRNA plasmid containing the GFP ORF and E. coli protein expression cassette. Enlarged, boxed nucleotides are the SD and DB motifs of the translation initiation sequence. Circular species show either the monomeric or polyGFP mRNA created after transcription and splicing. Translated portion of circular mRNAs is shown as double circle. The monomeric GFP-encoding mRNA, created after transcription and splicing, encodes a ∼30 kDa GFP species. The polyGFP-encoding mRNA has UAU in place of UAA termination codon, and is devoid of stop codons in the GFP reading frame. The initiating AUG (translation start) and fused 5′ss/3′ss (jagged arrowsplice junction) are shown on the circular mRNA species for both monomeric and polymeric constructs. Other abbreviations are: GFP, green fluorescent protein ORF; SD, Shine-Dalgarno sequence; AUG, initiating codon; DB, downstream box. (C) Same as (B), except circular GFP mRNA plasmid containing the GFP ORF and IRES for mammalian protein expression cassette. Linear species shows relevant portions of circular GFP mRNA plasmids including both GFP-AUG and GFP-stop mutations (see Subheading 3.1. ). The circular species show each GFP-encoding circular mRNA after transcription and splicing. The monomeric GFP-encoding mRNA contains a single UAA termination codon, as indicated by *, and encodes ∼50 kDa protein. The polyGFP-encoding species has a two base insertion at this position (indicated by triangle), and is devoid of stop codons in the GFP reading frame. “IRES” is the internal ribosome entry sequence required for ribosome recruitment (see Subheading 3.1.4. ).
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Ford, E. and Ares, M. (1994) Synthesis of circular RNA in bacteria and yeast using RNA cyclase ribozymes derived from a group I intron of phage T4. Proc. Natl. Acad. Sci. USA 91, 3117–3121.
Puttaraju, M. and Been, M. D. (1996) Circular ribozymes generated in Escherichia coli using group I self-splicing permuted intron-exon sequences. J. Biol. Chem. 271, 26,081–26,087.
Perriman, R. and Ares, M. (1998) Circular mRNA can direct translation of extremely long repeating sequence proteins in vivo. RNA 4, 1047–1054.
Long, M. B. and Sullenger, B. A. (1999) Evaluating group I intron catalytic efficiency in mammalian cells. Mol. Cell Biol. 19, 6479–6487.
Chen, C. Y. and Sarnow, P. (1995) Initiation of protein synthesis by the eukaryotic translational apparatus on circular RNAs. Science 268, 415–417.
Prince, J. T., McGrath, K. P., DiGirolamo, C. M., and Kaplan, D. L. (1995) Construction cloning and expression of synthetic genes encoding spider dragline silk.Biochemistry 34, 10,879–10,885.
Oshimi, Y. and Suzuki, Y. (1977). Cloning of the silk fibroin gene and its flanking sequences. Proc. Natl. Acad. Sci. USA 74, 5363–5367.
Sudo, S., Fujikawa, T., Nagakura, T., et al. (1997) Structures of mollusc shell framework proteins. Nature 387, 563–564.
Heslot, H. (1998) Artificial fibrous proteins: a review. Biochemie 80, 19–31.
Shine, J. and Dalgarno, L. (1974) The 3′-terminal sequence of Escherichia coli 16S ribosomal RNA: complementarity to nonsense triplets and ribosome binding sites. Proc. Natl. Acad. Sci. USA 71, 1342–1346.
Steitz, J. A. and Jakes, K. (1975) How ribosomes select initiator regions inmRNA: base pair formation between the 3′ terminus of 16S rRNA and the mRNA during initiation of protein synthesis in Escherichia coli. Proc. Natl. Acad. Sci. USA 72, 4734–738.
Gold, L. (1988) Posttranscriptional regulatory mechanisms in Escherichia coli.Annu. Rev. Biochem. 57, 199–233.
Sprengart, M. L., Fuchs, E., and Porter, A. G. (1996) The downstream box: an efficient and independent translation initiation signal in Escherichia coli. EMBO J. 15, 665–674.
Hellen, C. U. and Wimmer, E. (1995) Translation of encephalomyocarditis virus RNA by internal ribosomal entry. Curr. Top. Microbiol. Immunol. 203, 31–63.
Chalfie, M., Tu, Y., Euskirchen, G., Ward, W. W., and Prasher, D. C. (1994) Green fluorescent protein as a marker for gene expression. Science 263, 802–805.
Puttaraju, M. and Been, M. D. (1992) Group I permuted intron-exon (PIE) sequences self-splice to produce circular exons. Nucl. Acids Res. 20, 5357–5364.
Heim, R., Prasher, D. C., and Tsien, R. Y. 1994 Wavelength mutations and posttranslational autoxidation of green fluorescent protein. Proc. Natl. Acad. Sci. USA 91, 12,501–12,504.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2002 Humana Press Inc.
About this protocol
Cite this protocol
Perriman, R. (2002). Circular mRNA Encoding for Monomeric and Polymeric Green Fluorescent Protein. In: Hicks, B.W. (eds) Green Fluorescent Protein. Methods in Molecular Biology, vol 183. Humana Press. https://doi.org/10.1385/1-59259-280-5:069
Download citation
DOI: https://doi.org/10.1385/1-59259-280-5:069
Publisher Name: Humana Press
Print ISBN: 978-0-89603-905-6
Online ISBN: 978-1-59259-280-7
eBook Packages: Springer Protocols