Domain of E. coli DNA polymerase I showing sequence homology to T7 DNA polymerase

Escherichia coli contains three DNA polymerases that differ in their size, ability to interact with accessory proteins and biological function. Monomeric DNA polymerase I (Pol I) has a relative molecular mass (Mr) of 103,000 (103K) and is involved primarily in the repair of damaged DNA and the processing of Okazaki fragments; polymerase II is of Mr 120K, and polymerase III has a Mr of 140K, is responsible for the replication of the DNA chromosome and is just one of several proteins that are required for replication. DNA polymerases from bacteriophage as well as those of eukaryotic viral and cellular origin also differ with respect to their size and the number of associated proteins that are required for them to function in replication. However, the template-directed copying of DNA is identical in all cases. The crystal structure of the large proteolytic fragment of Pol I shows that it consists of two domains, the larger of which contains a deep crevice whose dimensions are such that it can bind duplex DNA. The T7 polymerase consists of two subunits, the 80K gene 5 protein and the host-encoded 12K thioredoxin of E. coli. We show here that there is an amino acid sequence homology between at least eight polypeptide segments that form the large cleft in the Klenow fragment and polypeptides in T7 DNA polymerase gene 5 protein, suggesting that this domain evolved from a common precursor. The parts of the Pol I and T7 DNA polymerase molecules that bind the DNA substrate appear to share common structural features, and these features may be shared by all of these varied DNA polymerases.     

Establishment of a coupled expression system mediated by modified T7 RNA polymerase gene

A coupled expression system for plants wasmerase gene was modified by addition of the coding sequenceof nuclear location signal from SV40 large T antigen. Plantexpression vector pBBT7 was constructed with the modifiedT7 RNA polymerase gene under the control of CaMV35Spromoter. Another expression vector pBTG contained cas-sette of gusA controlled by T7 promoter. The two vectorswere co-transformed into tobacco via the Agrobecte-rium-mediated method. Results of GUS activity indicatedthat the co-transformed plant with pBBT7 and pBTGshowed a high level of GUS activity. The results demon-strated that the coupled expression system of T7 polymeraseand T7 promoter was workable in plants.