For in vitro transcription from DNA templates, two classes of bacterial DNA- ... Finally, the template-independent poly(A) polymerase of E. coli is used to tail.
RNA Polymerases 1
UNIT 3.8 1
1
Beth M. Paschal, Larry A. McReynolds, Christopher J. Noren, and Nicole M. Nichols1 1
New England Biolabs, Ipswich, Massachusetts
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. C 2008 by John Wiley & Sons, Inc. Mol. Biol. 84:3.8.1-3.8.8. Keywords: in vitro transcription r RNA probes r RNAi r runoff transcription r RdRp r miRNA cloning
INTRODUCTION Commonly available RNA polymerases are derived from bacteria and bacteriophages and can be classified as DNA-dependent, RNA-dependent, or template-independent enzymes. For in vitro transcription from DNA templates, two classes of bacterial DNA-dependent RNA polymerases have been employed. One class is represented by E. coli RNA polymerase, a large multisubunit enzyme (∼480 kDa) that initiates transcription following recognition of the −10 and −35 sequence elements within a large (∼40 bp) promoter region. E. coli RNA polymerase was the first DNA-dependent RNA polymerase used for the synthesis of transcripts in vitro; however, it is problematic for many reasons including premature transcription termination in vitro (due to subunit dissociation) and the inability to generate a uniform population of RNA (due to the frequency of secondary initiation sites and the lack of a reliable way to direct precise termination of transcription at single-nucleotide resolution). Transcription protocols using a different class of DNA-dependent RNA polymerases became available in the 1980s and are now preferred over E. coli RNA polymerase: the bacteriophage T7, T3, and SP6 RNA polymerases (reviewed in Chamberlin and Ryan, 1982). These enzymes are the products of gene 1 from a family of related bacteriophages. Each is a single-subunit enzyme (∼95 to 98 kDa) that recognizes and initiates transcription specifically and exclusively from its own 18-bp promoter sequence. As discussed below, these properties have allowed the phage RNA polymerases to largely supplant E. coli RNA polymerase as the enzymes of choice for in vitro transcription. The RNA-dependent RNA polymerase (RdRp) from the bacteriophage phi6 can be used to generate dsRNA for RNA interference (RNAi) applications, resulting in in vivo gene silencing. Finally, the template-independent poly(A) polymerase of E. coli is used to tail or label RNA at the 3 terminus. Tailing RNA molecules that otherwise lack a poly(A) tail allows copying with oligo(dT) primers by reverse transcription and cloning of the Current Protocols in Molecular Biology 3.8.1-3.8.8, October 2008 Published online October 2008 in Wiley Interscience (www.interscience.wiley.com). DOI: 10.1002/0471142727.mb0308s84 C 2008 John Wiley & Sons, Inc. Copyright
Enzymatic Manipulation of DNA and RNA
3.8.1 Supplement 84
resulting cDNA. RNA molecules that are radioactively or fluorescently labeled using poly(A) polymerase can be used as probes in a wide variety of applications.
ENZYME: PHAGE RNA POLYMERASES: T7, T3, SP6 Phage T7, T3, and SP6 RNA polymerases are extremely processive enzymes used for high-yield transcription of DNA sequences inserted downstream from the corresponding T7, T3, or SP6 promoter. Transcripts thousands of nucleotides in length are readily obtained without the enzyme dissociating from the template. Transcription is both extensive and rapid, with elongation rates 10-fold higher than E. coli RNA polymerase, and RNA yields of up to 1 to 2 mg/ml can be readily obtained. Initiation is extremely specific for the individual promoter sequence, allowing the generation of labeled RNA probes that are strand specific. Plasmids have been constructed with polylinker cloning sites adjacent to the individual RNA polymerase promoters. Often the plasmid is cleaved with a restriction enzyme prior to transcription, resulting in the synthesis of “runoff” transcripts that terminate at the end of the DNA template (Fig. 3.8.1). In addition, the 5 and 3 ends of the transcript can be precisely defined by promoter placement and the distal end of the template strand, respectively. The phage polymerases have been cloned and are purified from overexpressing strains, resulting in inexpensive homogeneous enzyme preparations with extremely high specific activities.
Reaction Conditions Preparation of DNA template Because template design and preparation are crucial to the success of subsequent transcription reactions, the following should be considered: (1) DNA should contain an appropriately positioned T7 (5 -TAATACGACTCACTATAG), T3 (5 -AATTAACCCTCACTAAAG), or SP6 (5 -ATTTAGGTGACACTATAG) promoter
5′
Cloned DNA
5′ T7 promoter
RNA transcripts
X 5′ 5′ 5′
Vector DNA digested with restriction enzyme X
X
3′
5′ 3′ T7 RNA polymerase
5′ 3′
X 5′
T7 promoter Vector DNA
Cloned DNA Template
RNA Polymerases
Figure 3.8.1
Runoff transcripts.
3.8.2 Supplement 84
Current Protocols in Molecular Biology
(underlined base corresponds to the 5 base of the resulting RNA transcript). (2) Plasmids purified using standard miniprep procedures (UNIT 1.6) or commercially available kits are generally contaminated with RNase A. If using miniprep plasmid DNA as a template, RNase inhibitor (UNIT 3.13) MUST be added to the transcription reaction. (3) For runoff transcription of plasmid templates, the restriction enzyme used for linearization should yield either a 5 overhang or a blunt end. Linearization of a template with an enzyme that produces a 3 overhang will result in aberrant transcripts (Schenborn and Mierendorf, 1985). DNA should be purified from restriction digestion reactions by phenol/chloroform extraction and ethanol precipitation (UNIT 2.1A) or using commercial spin columns (e.g., QIAGEN QIAquick). (4) Alternatively, linear DNA templates can be prepared by PCR. The promoter sequence can be included in the upstream PCR primer if it is not present in the initial PCR template. The 5 end of this primer can correspond to the 5 end of the promoter sequences listed above. To avoid templates with 3 overhangs, nonproofreading polymerases (e.g., Taq polymerase) should not be used for PCR; instead proofreading polymerases (such as Pfu, Vent, and Phusion) are preferred. (5) For short RNA transcripts (
