Transcription of T7 DNA

Bacteriophage T7 depends initially on the transcriptional machinery of the host. Transcription of T7 DNA in vitro should, therefore, imitate the first steps of infection. 

In vitro transcription of T7 DNA by purified E, coli RNA pol5unerase in the absence of additional factors yields a specific RNA product (Millette et ah, 1970 confirmed by Maitra et ah, 1970, and Dunn and Studier, 1973). The majority of this RNA has an approximate molecular weight of 2.4 X 10 daltons a small amount of 3.4 X 10 dalton species is also synthesized (Millette et ah, 1970). End labeling experiments (Kamen, I969 Leppla et ah, 1968) indicate that the 2.4 X10 dalton species is terminated uniquely with U (Millette et ah, 1970) and the 3 -terminal decanucleotide has been identified (Millette et ah, 1970). Thus, in vitro, E. coli RNA polymerase transcribes this RNA species from one promoter and terminates at a distinct point without the need for any additional factors. 

Infection of cells with DNA phage may lead to the production of a new, phage-spedfic RNA-P. (e.g. T7 phage RNA-P. is a si e-chtiin protein, M, 107,000), or the host RNA-P. may be modfied with the addition of new protein subunits encoded by the phage (e.g. T4 and k phage) in each case the resulting RNA-P. is specific for transcription of phage DNA. 

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. 

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. 

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...

Williamson, P. and Felsenfeld, G. (1978) Transcription of histone-covered T7 DNA by Escherichia coli RNA polymerase. Biochemistry 17, 5695-5705. 

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. 

Table 11.5 reports also the work by Fischer et al. (2002) on m-RNA synthesis inside giant vesicles utihzing a DNA template and T7 RNA polymerase and the transcription of DNA hy Tsumoto et al. (2002). 

Excessive amounts of RNA polymerase should not be used with the vector containing both T3 and T7 promoters. Otherwise, transcription may not be promoter-specific (strand-specific) Nonspecific initiation of RNA transcripts may also occur at the ends of the DNA template. This is most prevalent with a 3 -protrud-mg terminus Nonspecific initiation may be reduced by increasing the final NaCl concentration in the transcription buffer to 100 mM. When possible, restriction enzymes that leave blunt or 5 -protruding ends should be used... 

Fig. 8.2. Design of a syn tire tic DNA random library template for use as starting material for in vitro selection. Various sites are engineered into this construct to allow for PCR by Taq polymerase, transcription by T7 RNA polymerase, and ligation with T4 DNA ligase. This construct also includes a 100-nucleotide region of random sequence that will become the evolved catalytic region.

The second assay makes use of the isothermal self-sustained sequence replication reaction of RNA (3SR Fahy et al., 1991). Instead of double strand melting to yield single strands the RNA DNA hybrid obtained through reverse transcription is converted into single stranded DNA by RNA digestion making use of RNase H. DNA double strand synthesis and transcription complete the cycle. Here, transcription by T7 polymerase represents the amplification step. 

Fig. S. In vitro transcription of a cloned DNA fragment from Anabaena azollae containing a self-splicing intron. (A) Restriction map of a 2.7-kb insert in pBSM13—. Arrows indicate direction of transcription from the T3 and T7 promoters in the vector. tRNA exon (solid) and intron (hatched) sequences are indicated. (B) Plasmid DNA truncated by Ps/I (P), SlpI (S), Dral (D), and Hindi) (H) (in the 3 exon), downstream of the T3 promoter. After transcription with T3 RNA polymerase, products were fractionated on a 3% polyacrylamide-8 M urea gel the autoradiogram is shown. Scale at left indicates position of Haelll restriction fragments of phage 0X174 DNA in nucleotides. Labels indicate positions expected for the unspliced run-off transcript (Pre), ligated exons (LE), and linear intron (LI). (From Xu et at. Copyright 1990 by the AAAS.)...

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