T7 polymerase

The RTS system includes two different technology platforms for cell-free protein expression as well as a number of tools for finding optimal conditions (Scheme 1.1). All expression systems use the T7-polymerase for transcription and an E. coli lyzate with reduced nuclease and protease activity for translation. The conditions are optimized for a coupled transcription/translation reaction so that the DNA can be directly used as the template. 

BL21-Ai BL21 T7 polymerase gene present under the control of the araBAD promoter leads to much tighter arabinose-inducible expression... 

LysS/LysE an additional chloramphenicol resistant plasmid carries the gene forT7 lysozyme under constitutively active promoters. T7 lysozyme inhibits the activity of T7 polymerase thereby reducing basal (uninduced) polymerase activity/ protein expression. LysE express higher levels of T7 lysozyme for tighter control. pLacI an additional chloramphenicol resistant plasmid carries the gene for high level production of the lac repressor to reduce basal expression. 

Pol. mini-Tn5 transposon using lacZ blue/white screen, then delivering the transposon to stable insertion in host chromosome. Another mini-Tn5 provides T7 polymerase inducible by benzoate. Seven different antibiotic selections available to allow multiple genes expressed in same host. ... 

Some bacteriophage encode their own DNA polymerases. However, they usually rely on the host cell to provide accessory proteins. The sequence of the DNA polymerase from phage T7 is closely homologous to that of the Klenow fragment and the 3D structures are similar. The 80-kDa T7 polymerase requires the 12-kDa thioredoxin from the host cell as an additional subunit. It has been genetically engineered to improve its usefulness in DNA sequencing 278... 

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. 

Studier, F. W. and Moffatt, B. A. (1986) Use of bacteriophage T7 polymerase to direct selective high-level expression of cloned genes. J. Mol. Biol. 189, 113-130. 

As already noted, templated sequencing protocols are now the most generally used methods for nucleic acid sequence determination. The use of the highly processive modified bacteriophage T7 polymerase (Sequen-ase U.S. Biochemical Corp., Cleveland, OH) has eliminated many earlier problems associated with variable intensity of individual bands on sequencing gels. 

T7 polymerase will read the top (template) strand 3 — -5 and produce a transcript in the 5 — -3 direction that is identical to the bottom (coding) strand, with uracil (U) in place of thymine (T). 

Antisense RNA amphfication (aRNA) Target T4 DNA polymerase Klenow SI nuclease T7 polymerase No... 

For optimal transcriptional efficiency of T7 polymerase a few points should be considered. The initial base in the transcript must be a purine and preferably a G (the optimal sequence is (5 -Gp(G/C)).8 However, the polymerases accept a wide range of modifications at the 5 positions such as GpppG, dinucleotides and biotinylated dinucleotides (provided the 3 -nucleotide is a G, Section 2.3.2). If transcriptions are performed under conditions where one of the nucleotides is limiting the sequence of the first ten nucleotides is crucial for a high yield. If [a-32P] UTP is limiting in cotranscriptional labelling of RNA uridines within the initial ten nucleotides of the transcript will result in a high frequency of short abortive transcripts. This has been rationalised as a result of a transition from a labile initiation complex (the initial ten nucleo-... 

Phosphate modifications T7 polymerase does not discriminate significantly between NTP and [aS] NTP (NEN). To obtain the appropriate modification level the NTP is substituted with [aS] NTP to the desired level. A standard transcription protocol, such as the one described in Section 2.3.1, can be used. 

Milligan, J.F. and Uhlenbeck, O.C. (1989). Synthesis of small RNAs using T7 polymerase. Meth. Enzymol. 180, 51-62. 

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