Cell-free Transcription and Translation

In Vitro Translation Systems Researchers synthesize recombinant proteins in cell-free extracts to verify the identity of cloned genes, to study protein-protein, protein-nucleic acid, and protein-drug interactions, and to carry out mutagenesis studies. In vitro protein translation studies rely on the efficient and selective transcription of cloned genes in vitro, which is now not only possible, but also routine, thanks to the identification of bacteriophage (phage) DNA-dependent RNA polymerases and their promoters.1 Phage RNA polymerases are single subunit enzymes that polymerize RNA from DNA templates at rates up to five times faster than bacterial polymerases. Three phage RNA polymerases are generally used for in vitro transcription: Salmonella typhimurium phage SP6 RNA polymerase, Escherichia coli phage T3 RNA polymerase, and E. coli phage T7 RNA polymerase.1 These three polymerases are highly specific for their individual promoters, recognize promoter sequences not likely to occur in mammalian or bacterial DNA sequences, and can produce full-length transcripts of genes placed under the control of their individual promoters.2 Phage polymerases can also transcribe DNA templates with a high degree of secondary structure. In 1984, Melton and colleagues first generated runoff transcripts with SP6 RNA polymerase in vitro.3 Since that time, researchers have also used T3 and T7 promoters extensively for in vitro transcription. Due to their short sequence length, these phage promoters can be incorporated into PCR primers to transcribe amplified DNA. Transcription and translation reactions may be either linked or coupled. In linked translation systems, transcription and translation occur as separate reactions. RNA is first transcribed, and then a small fraction of the transcription reaction is used to drive translation. In contrast, coupled transcription and translation systems perform both reactions in a single tube. Since both transcription and translation reactions can be optimized in linked systems, they generally produce increased yields of translated protein when compared to coupled systems. Courtesy of AmbionLinked transcription and translation reactions In vitro translation Cell-free translation systems are valuable protein analysis tools that offer a number of benefits. They can direct protein synthesis from a single DNA or RNA species under controlled conditions without the background synthesis that normally occurs from endogenous mRNA in vivo. In addition, proteins synthesized in cell-free systems can be labeled to monitor their interactions and biological activities. Fusion of the translated protein to a purification tag can be used to quickly isolate the protein from an in vitro translation reaction. Finally, in vitro translation systems can be used to synthesize proteins when an overexpressed gene product is toxic to the host cell, when the gene product is insoluble, or when the protein is rapidly degraded by intracellular proteases. The identity of a cloned gene or PCR fragment can be determined after in vitro translation by using specific antibodies in Western blotting or immunoprecipitation assays. Alternatively, if the translated protein has measurable biological activity, then this activity can be used to definitively identify the gene product. The size of the protein can be measured by direct staining of SDS-polyacrylamide gels, or by detection of incorporated labels, such as 35S-methionine, biotinylated lysine, and BODIPY(r)-labeled lysine (Fluorotect(tm) GreenLys from Promega of Madison, Wis.). Protein-protein interactions can also be examined using in vitro translated proteins. Proteins that may interact in vivo can be translated in vitro, either separately or in a single tube, immunoprecipitated with an antibody that recognizes one of the two proteins, and identified by SDS-PAGE or Western blotting. Toleg Osipovich and colleagues at the National Institutes of Health, recently examined subunit interactions of the Ku protein by in vitro translation.4 Ku is a heterodimer of 70 and 80 kDa subunits that is active in the repair of double-stranded DNA breaks. It is also a major autoantigen expressed in patients suffering from autoimmune diseases. Specific mutations were created within the Ku80 gene that inhibited the ability of this subunit to interact with the 70 kDa subunit, thus identifying the minimal amino acid domain of Ku80 required for interaction with Ku70. Interactions of proteins and nucleic acids (e.g. transcription factor-DNA interactions) can be assessed using in vitro translated proteins in electrophoretic mobility shift assays.5 By translating the candidate protein in vitro and incubating it with an oligonucleotide to identify changes in the oligo's mobility, these "gel-shift" assays can be used to identify both protein-DNA and protein-RNA interactions. Finally, in vitro translation can be used to identify mutations that affect protein size, such as premature termination and frameshift mutations. For example, large gene families often contain a large number of nonfunctional pseudogenes (e.g., the human olfactory receptor family).6 Recently, a family of bitter taste receptor genes that may also contain nonfunctional pseudogenes was identified in humans.7 Use of in vitro translation would be one means to screen these two gene families for nonfunctional members. Maximizing Efficiency A prokaryotic or eukaryotic ribosome-binding site must be present on the RNA template upstream from the coding sequence to efficiently translate proteins in vitro. In bacteria, the ribosome binds to the mRNA at the Shine-Dalgarno (SD) sequence, a purine-rich sequence, 8-11 bases long, that is complementary to the 3' end of the 30S ribosomal subunit 16S RNA. The position of the SD sequence relative to the AUG start codon is critical for efficient translation: it is located 6-10 bases upstream of the first base of the initiation codon. Most eukaryotic mRNA molecules contain a 7-methylguanylate (7mG) cap at the 5' end of the message that is formed by a 5'-5' pyrophosphate linkage to the first nucleotide of the mRNA; this cap is absent in prokaryotic transcripts. The 5' cap is important for binding of translation initiation factors to the mRNA, and it may contribute to mRNA stability; a 7mG cap can be directly incorporated into RNA transcribed in vitro by using a 7mG analog. These analogs are available from a number of vendors. Capped RNA is generally required for efficient translation in cell-free extracts from rabbit reticulocyte or wheat germ translation systems. Eukaryotic ribosomes are thought to initiate translation by a scanning mechanism in which the 40S ribosomal subunit, initiator methionine-tRNA complex, and initiation factors, bind to the capped 5' end of the mRNA molecule.8 A consensus sequence that is thought to cause the 40S ribosomal subunit to pause and join with the 60S ribosomal subunit, has been identified just 5' to the AUG start codon in eukaryotic mRNA. This Kozak consensus sequence spans nucleotide positions -6 to +4 of vertebrate mRNA.8 A purine at position -3 (preferably an A) and a G at nucleotide +4 generally yield a strong translation initiation site. Cell-free translation extracts In vitro translation components can be prepared from either prokaryotic or eukary-otic sources,5 and can be programmed with either cellular or in vitro transcribed RNA. The most widely used cell-free translation systems utilize extracts from rabbit reticulocytes, wheat germ, or E. coli. These extracts can be treated with nucleases to destroy endogenous mRNAs. They contain the two ribosomal subunits, transfer RNAs, aminoacyl-tRNA synthetases, and necessary initiation, elongation, and termination factors. Energy sources such as ATP or GTP, along with a eukaryotic or prokaryotic energy regenerating system, are required to drive translation. Reticulocytes are specialized nonnucleated blood cells that synthesize hemoglobin and other erythrocyte proteins. Rabbit reticulocyte lysates (RRL) are primarily used to translate large mRNA templates, and they often carry out posttranslational processing activities such as proteolysis, acetylation, and phosphorylation. Canine pancreatic microsomal membranes can be added to RRL to drive cotranslational processing events such as signal peptide cleavage with a signal peptidase, glycosylation, or membrane insertion. One potential drawback with reticulocyte systems is that they are inhibited by oxidized thiols, low concentrations of double-stranded RNA, and polysaccharides. In addition, reticulocyte lysates are only efficient over a narrow magnesium ion concentration range. Promega's TNT(r) Quick Coupled Transcription/ Translation System is a single tube RRL system designed for translation of eukaryotic genes cloned downstream from either an SP6 or a T7 promoter within circular plasmids, or from PCR-generated DNA fragments with an upstream T7 promoter. Ambion of Austin, Texas, offers the PROTEINscript(tm) II linked transcription-translation kit, which is useful for efficient in vitro synthesis of proteins from PCR products, supercoiled DNA, or linear DNA. Product manager Jessica Gott reports that the PROTEINscript II(tm) system utilizes an RRL that has been optimized for the efficient translation of eukaryotic and prokaryotic mRNAs. A choice of transcription mixes containing SP6, T3, or T7 RNA polymerase is available, typically yielding 50-200 nanograms of translated protein. Novagen of Madison, Wis., also offers the Single Tube Protein(r) System 3 (STP3), a linked RRL-based translation system designed for rapid and efficient in vitro translation of proteins from supercoiled DNA, linear DNA, or PCR-generated fragments. Transcription can be driven from either a T7 or SP6 promoter. Biotinylated proteins can be produced using Novagen's STP3-biotin kit. A second cell-free system is derived from wheat germ (the embryo plant within a fertile wheat seed). Wheat germ extracts (WGE) contain the essential cellular components for in vitro synthesis of proteins, but endogenous amino acids, mRNAs, and plant pigments are usually removed prior to use. Because they lack the small globin proteins found in RRL, WGE are preferred for synthesizing small proteins; they also exhibit low background protein synthesis because relatively few endogenous mRNAs remain in the lysate. WGE can efficiently translate both RNA transcripts prepared in vitro and purified cellular poly A+ RNA, and they can use RNA templates that contain contaminants that generally inhibit translation in RRL.9 However, wheat germ extracts are more cap-dependent than are reticulocyte systems, and RRL appear to be better at making post-translational modifications than are wheat germ extracts. Commercial WGE systems are available from Ambion, Promega, and Roche Molecular Biologicals of Indianapolis. Since transcription and translation are coupled processes in bacteria, E. coli cell-free extracts are useful for coupled transcription and translation from DNA templates using either a phage or bacterial promoter. They are not generally suitable for translation of exogenous cellular RNA because these extracts contain endogenous nucleases. Because bacterial extracts do not require capped template RNAs, both prokaryotic and eukaryotic gene sequences can be efficiently translated in cell-free bacterial systems. However, prokaryotic cell-free systems do not generally catalyze the post-translational modification of proteins common in eukaryotic systems. Bacterial extracts have a long pedigree. They were used to translate nucleotide homopolymers for elucidation of the genetic code, and the first protein to be completely translated in a cell-free system (phage f2 coat protein) was produced in a bacterial extract. These S30 bacterial extracts are still used today for in vitro translation studies10 and to incorporate nonstandard amino acids into proteins for subsequent structural studies.11 Promega offers an E. coli T7 S30 Extract System for circular DNA templates that is ideal for translation of DNA sequences that contain a T7 promoter. Ambion's PROTEINscript PRO(tm) also uses E. coli S30 extracts, but it can utilize circular or linear DNA templates that contain either a phage promoter or an endogenous E. coli promoter. Proteins up to 60 kDa is size (about 550 amino acids) can be efficiently translated, and yields of 100 to 1000 nanograms of protein per reaction can be obtained. Novagen's recently introduced EcoPro(tm) System uses fractionated EcoPro and EcoPro T7 extracts to synthesize full-length proteins from genes downstream from E. coli and T7 promoters on circular or linear DNA templates. The production of secondary products is significantly reduced with EcoPro when compared to traditional S30 extracts. Though in vitro translation systems typically produce small yields, Roche Molecular Biochemicals recently introduced the Rapid Translation System RTS 500 for preparative-scale protein expression. This system employs instrumentation that incorporates a patented continuous-exchange cell-free technology, continually removing the inhibitory products that may accumulate during protein synthesis, and continuously replacing required substrates and energy sources. A single reaction can produce more than 500 µg of product, and proteins greater than 120 kDa in size have been translated with this system. The system is especially suited for cytotoxic proteins that cannot be expressed in traditional cell-based expression systems. Gregory Smutzer (smutzer@hotmail.com) is a freelance writer in Lansdowne, Pa. References 1. E.T. Butler, M.J. Chamberlin, "Bacteriophage SP6-specific RNA polymerase I. Isolation and characterization of the enzyme," Journal of Biological Chemistry, 257:5772-8, 1982. 2. F.W. Studier et al., "Use of T7 RNA polymerase to direct expression of cloned genes," Methods in Enzymology, 185:60-89, 1990. 3. D.A. Melton et al., "Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter," Nucleic Acids Research, 12:7035-56, 1984. 4. O. Osipovich et al., "Defining the minimal domain of Ku80 for interaction with Ku70," Journal of Biological Chemistry, 272:27259-65, 1997. 5. "Protein translation in vitro," in Protocols and Applications Guide, 3rd edition (K. Doyle, ed). Madison, Wis., Promega Corp., pages 259-88, 1996. 6. S. Rouquier et al., "Distribution of olfactory receptor genes in the human genome," Nature Genetics 18:243-50, 1998. 7. E. Adler et al., "A novel family of mammalian taste receptors," Cell, 100:693-702, March 17, 2000. 8. M. Kozak, "An analysis of vertebrate mRNA sequences: Intimations of translational control," Journal of Cell Biology, 115:887-903, 1991. 9. D. Wilkinson, "Cell-free and happy: In vitro translation and transcription/translation systems," The Scientist, 13[13]:15, June 21, 1999. 10. G. Zubay, "In vitro synthesis of protein in microbial systems," Annual Review of Genetics, 7:267-87, 1973. 11. C.J. Noren et al., "A general method for site-specific incorporation of unnatural amino acids into proteins," Science, 244:182-8, 1989.

Cell-free Transcription and Translation

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