T7 RNA polymerase

Like SP6 and T3 RNA polymerases, T7 RNA Pol displays a stringent specificity for its own promoter to start transcription (1—3). The consensus promoter sequence derived from all 17 T7 promoters is a highly conserved sequence of 23 continuous base pairs (Fig. 7.3). The nucleotide positions from —7 to +1 are highly conserved in all three phage promoters. The promoter sequences differ significantly in the region from —8 to —12, suggesting that the base pairs in 

To protect RNA transcripts from possible degradation by contaminating RNases, the RNase inhibitor (RNasin) may be added at 1 U//il final concentration. For the synthesis of capped transcripts, the GTP concentration is reduced to one tenth that of the noncapping reaction while G(5 )ppp(5 )G or its methylated cap analog is added at a cap analog/GTP ratio of 4. The reaction is stopped by adding EDTA to 50 mM or by heating to 65°C for 10 min. 

For plasmid DNA templates, the yield can be over 30 /tg RNA transcripts per fig of template DNA under the recommended conditions and as high as 200 fig RNA under some high-yield conditions. The DNA template usually does not interfere with subsequent applications of the RNA transcripts. If the removal of the DNA is desired, the reaction mixture can be digested with 20 figJmX RNase-free DNase for 15 min at 37°C and the DNase removed by phenol/chloroform extraction. Alternatively, the reaction mixture is passed through a Mono Q column to purify RNA transcripts as well as to recycle the DNA template (8). 

Chloride ion is presumed to bind, as does the phosphate ion, to the anionbinding site of the enzyme in competition with the backbone phosphates of substrate DNA. In fact, the apparent K, but not is strongly dependent on the concentration of NaCl the increases over 20-fold when [NaCl] increases from 10 to 100 mM (105). In contrast to NaCl or KCl, T7 RNA Pol well tolerates potassium glutamate (KGlu), at least up to 100 mM. 

Chemicals and biologicals DTT or 2-MSH significantly stabiUzes some preparations of T7 RNA Pol. (Note For long-term storage of the enzyme, the addition of DTT is recommended every 6 months.) The enzyme is inactivated by SH reagents such as lAA, iodoacetamide, and PCMB and also by air oxidation. 

---

As seen in the genetic map, the genes after gene 1.1, transcribed by the T7 RNA polymerase, code for proteins that are involved in T7 DNA synthesis, the formation of virus coat proteins, and assembly. Three classes of T7 proteins are formed class I, made 4-8 minutes after infection, which use the cell RNA polymerase class II, made 6-15 minutes after infection, which are made from T7 RNA polymerase and are involved in DNA metabolism class III, made from 6 minutes to lysis, which are transcribed by T7 RNA polymerase and which code for phage assembly and coat protein. This sort of sequential pattern, commonly seen in many large double-stranded DNA phages, results in an efficient channeling of host resources, first toward DNA metabolism and replication, then on to formation of virus particles and release of virus by cell lysis. 

Na salts of ribonucleotide triphosphates (Roche or Sigma) bovine serum albumin RNase-free, 20 mg/ml (Roche) RNasin ribonuclease inhibitor, 40 U/ml (Promega) both bacteriophage T7 RNA polymerase and RNA Cap structure analog m7G(5/)ppp(5/)G are from BioLabs DNase-RNase-free (Roche) complete EDTA-free proteinase inhibitors cocktail (Roche) pyruvate kinase (PK) (Roche). 

Because the yield of transcription can vary depending upon a large number of factors (type and quality of the DNA template, T7 RNA polymerase, ribonucleotide triphosphates, etc.), it is recommended to optimize the reaction conditions on a small scale before embarking on a large-scale mRNA prep. 

Anion exchange chromatography The reaction mixture is subjected to phenol/chloroform extraction to remove the T7 RNA polymerase using phenol equilibrated with 50 mM Na acetate (pH 4.5). After isopropanol precipitation, the pellets are resuspended in 20 mM MOPS buffer (pH 6.25) containing 350 mM NaCl. The excess unincorporated NTPs and the smaller abortive transcription products are removed by chromatography on anion exchange FPLC column (MonoQ 5/5 column, Amersham). 

RNA Polymerases T7 RNA polymerase (New England Biolabs) and T3 RNA polymerase (Invitrogen). 

Ma, K., Temiakov, D., Jiang, M., Anikin, M., and McAllister, W.T. (2002) Major conformational changes occur during the transition from an initiation complex to an elongation complex by T7 RNA polymerase./. Biol. Chem. 277, 43206-43215. 

Mukherjee, S., Brieba, L.G., and Sousa, R. (2002) Structural transitions mediating transcription initiation by T7 RNA polymerase. Cell 110(1), 81-91. 

An amplification reaction that is used to amplify target RNA or denatured DNA is called the transcription-based amplification system (TAS). This technique involves using an enzyme called reverse transcriptase and a primer with sequence complementary to the sample target RNA molecule in order to synthesize a complementary DNA (cDNA) copy of the sample target RNA. After denaturation to separate the strands, another primer and additional reverse transcriptase are added to synthesize a double-stranded cDNA molecule. Since the first primer has also an RNA polymerase binding site, it can, in the presence of T7 RNA polymerase, amplify the double-stranded cDNA to produce 10 to 100 copies of RNA. The cycle of denaturation, synthesis of cDNA, and amplification to produce multiple RNA copies is repeated. With as few as four cycles, a 2- to 5-millionfold amplification of the original sample RNA target is possible. However, the time required to achieve a millionfold amplification is approximately 4 hours, which is the same amount of time required by PCR. The TAS requires, however, the addition of two enzymes at each cycle and, as such, can be cumbersome. 

Geib S, Sandoz G, Carlier E, Cornet V, Cheynet-Sauvion V, De Waard M. 2001. A novel Xenopus oocyte e3q>ression system based on cytoplasmic coinjection of T7-driven plasmids and purified T7-RNA polymerase. Receptors Channels 7 (5) 331. 

Schenborn ET, Mierendorf RC. 1985. A novel transcription property of SP6 and T7 RNA polymerases Dependence on template structure. Nucl Acids Res 13 6223. 

The suppressor tRNA developed by the Chamberlin lab for use in a rabbit reticulocyte lysate is based on an E. coli glycyl tRNA, which was initially chosen because glycyl-tRNA synthetases do not rely on a double-sieve editing mechanism for enzymatic hydrolysis of misacylated tRNAs [26]. Two base pair changes were made to the acceptor stem to allow incorporation of the optimal T7 RNA polymerase promoter into the DNA template for tRNA y-Con [27,28],... 

Building on earlier work of Osawa and co-workers [55], Oliver and Kowal [52] tested the feasibility of introducing a noncoded amino acid at an unassigned codon in M. luteus. DNA templates were prepared which coded for 19-mer polypeptides containing either the unassigned codon AGA(Arg) or the termination codon TAG at position 13 under the control of a T7 RNA polymerase promoter. The corresponding tRNAs, produced as described in Sect. 2, were based on tRNA and acylated with phenylalanine. The tRNA was modified to prevent recognition by the alanine aminoacyl-tRNA synthetase and to increase translational efficiency. 

Fig. 5. Selection scheme for the in vitro selection of RNA libraries. The RNA library is subjected to a selection criterion suitable for the enrichment of functionally active sequences. The few selected individual sequences are amplified by reverse transcription (RT) and polymerase chain reaction (PCR). The PCR-DNA is then subjected to in vitro transcription with T7 RNA polymerase. The resulting enriched and amplified RNA library can be used as the input for the next selection cycle. This process is repeated until active sequences dominate the library. At this point, individual sequences can be obtained by cloning and their sequence can be determined by sequencing...

DNA, since proximity eflfects demand that the DNA or nascent RNA closest to the histones at the point of disruption will be the polyanion for which those histones will preferentially reassociate. Ten Heggeler-Bordier et al. [95] have verified these observations. They used immuno-electron microscopy to determine what happens to histones after transcription with T7 RNA polymerase of a multi-nucleosomal template and also observed transfer to the nascent RNA. In contrast, Kirov et al. [96] have reported that no histones displace during transcription with this polymerase. However, as described above, transcriptional efficiency and ultimately histone displacement is not efficient in very low ionic strength conditions. 

The choice of vectors obviously depends upon the expression system that will be used and there is a wide availability of optimized reagents from both commercial and academic sources. For expression in E. coli, the most widely used system for HTP protein production is the pET/T7 promoter vector developed by Studier which makes use of T7 RNA polymerase to direct expression of cloned... 

Schenk PM, Baumann S, Mattes R, Steinbiss HH. (1995) Improved high-level expression system for eukaryotic genes in Escherichia coli using T7 RNA polymerase and rare ArgtRNAs. Biotechniques 19, 196-8, 200. 

Based on the initial report on the expression of functional protein into liposomes by Yomo and coworkers (Yu et al, 2001), the work by Ishikawa et al. (2004) represents another stage of the work on GFP expression. In fact, a two-stage genetic network is described, where the first stage is the production of T7 RNA polymerase, required to drive the GFP synthesis as the second stage. 

---