RNA Polymerases

Beth M. Paschal1, Larry A. McReynolds1, Christopher J. Noren1, Nicole M. Nichols1

1 New England Biolabs, Ipswich, Massachusetts
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 3.8
DOI:  10.1002/0471142727.mb0308s84
Online Posting Date:  October, 2008
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Abstract

This unit describes DNA‐dependent, RNA‐dependent, and template‐independent RNA polymerases. DNA‐dependent RNA polymerases include the related bacteriophage T7, T3, and SP6 polymerases, the most commonly used RNA polymerases for in vitro transcription reactions. Reaction conditions to produce preparative quantities of transcribed RNA and labeled RNA probes are covered, as are the major applications of these reactions. Limitations of the E. coli RNA polymerase for these applications are also presented. The properties of the phi6 RNA‐dependent RNA polymerase (RdRp) and its use in RNAi experiments are also introduced. Poly(A) polymerase, a template‐independent polymerase, catalyzes the incorporation of AMP residues onto the free 3′‐hydroxyl terminus of RNA, utilizing ATP as a precursor. Specific reaction conditions of poly(A) polymerase, as well as applications including RNA tailing and 3′ end labeling, are discussed. Curr. Protoc. Mol. Biol. 84:3.8.1‐3.8.8. © 2008 by John Wiley & Sons, Inc.

Keywords: in vitro transcription; RNA probes; RNAi; runoff transcription; RdRp; miRNA cloning

     
 
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Table of Contents

  • Introduction
  • Enzyme: Phage RNA Polymerases: T7, T3, SP6
  • Enzyme: Escherichia coli RNA Polymerase
  • Enzyme: Poly(A) Polymerase
  • Enzyme: phi6 RNA‐Dependent RNA Polymerase
  • Literature Cited
  • Figures
     
 
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Materials

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Figures

Videos

Literature Cited

   Chamberlin, M.J. and Ryan, T. 1982. Bacteriophage DNA‐dependent RNA polymerases. In The Enzymes, Vol. 15 (P.D. Boyer, ed.) pp. 87‐109, Academic Press, New York.
   Edmonds, M. 1982. Poly(A) adding enzymes. In The Enzymes, Vol. 15B (P.D. Boyer, ed.) pp. 218‐245. Academic Press, New York.
   Endo, Y. and Sawasaki, T. 2005. Advances in genome‐wide protein expression using the wheat germ cell‐free system. Methods Mol. Biol. 310:145‐67.
   Evans, P.D., Cook, S.N., Riggs, P.D., and Noren, C.J. 1995. LITMUS: Multipurpose cloning vectors with a novel system for bidirectional in vitro transcription. Biotechniques 19:130‐135.
   Gething, M.J., Bye, J., Skehel, J., and Waterfield, M. 1980. Cloning and DNA sequence of double‐stranded haemagglutinin genes from H2 and H3 strains elucidates antigenic shift and drift in human influenza virus. Nature (London) 287:301‐306.
   Lingner, J. and Keller, W. 1993. 3′‐End labeling of RNA with recombinant yeast poly(A) polymerase. Nucleic Acids Res. 21:2917‐2920.
   Makeyev, E.V. and Bamford, D.H. 2000. The polymerase subunit of a dsRNA virus plays a central role in the regulation of viral RNA metabolism. EMBO J. 19:6275‐6284.
   Martin, G. and Keller, W. 1998. Tailing and 3′‐end labeling of RNA with yeast poly(A) polymerase and various nucleotides. RNA 4:226‐230.
   Melton, D.A., Krieg, P.A., Rebagliati, M.R., Maniatis, T., Zinn, K., and Green, M.R. 1984. Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. Nucleic Acids Res. 12:7035‐7056.
   Milligan, J.F., Groebe, D.R., Witherell, G.W., and Uhlenbeck, O.C. 1987. Oligoribonucleotide synthesis using T7 RNA polymerase and synthetic DNA templates. Nucleic Acids Res. 15:8783‐8798.
   Sampson, J.R. and Uhlenbeck, O.C. 1988. Biochemical and physical characterization of an unmodified yeast phenylalanine transfer RNA transcribed in vitro. Proc. Natl. Acad. Sci. U.S.A. 85:1033‐1037.
   Schenborn, E.T. and Mierendorf, R.C. Jr. 1985. A novel transcription property of SP6 and T7 RNA polymerases: Dependence on template structure. Nucleic Acids Res. 13:6223‐6236.
   Shingara, J., Keiger, K., Shelton, J., Laosinchai‐Wolf, W., Powers, P., Conrad, R., Brown, D., and Labourier, E. 2005. An optimized isolation and labeling platform for accurate microRNA expression profiling. RNA 11:1461‐1470.
   Studier, F.W. and Moffatt, B.A. 1986. Use of bacteriophage T7 RNA polymerase to direct selective high‐level expression of cloned genes. J. Mol. Biol. 189:113‐130.
   Su, G., Li, H., and Rossi, J.J. 2007. Cloning and detecting signature microRNAs from mammalian cells. Methods Enzymol. 427:123‐138.
   Tabor, S. and Richardson, C.C. 1985. A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes. Proc. Natl. Acad. Sci. U.S.A. 82:1074‐1078.
   van der Horst, G. and Tabak, H.F. 1985. Self‐splicing of yeast mitochondrial ribosomal and messenger RNA precursors. Cell 40:759‐766.
Key Reference
   Chamberlin and Ryan, 1982. See above.
  Summarizes properties of DNA‐dependent bacterial phage RNA polymerases (T3, T7, and Sp6).
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