Affinity modification of DNA-dependent RNA-polymerase of phage T7 with 5'-p-fluorosulfonylbenzoyladenosine

The affinity modification of the DNA-dependent RNA-polymerase of bacteriophage T7 was carried out by using the specific irreversible inhibitor, 5'-p-fluorosulfonylbenzoyladenosine. The inhibitor was found to bind to the enzyme's active site; the kinetic constants of the modification were calculated. The stoichiometry of the covalent E.I-complex formed was determined by using the 14C-labeled inhibitor.     

Preparation and characteristics of monoclonal antibodies to DNA-dependent RNA-polymerase of bacteriophage T7

The monoclonal antibodies to DNA-dependent RNA-polymerase of bacteriophage T7 have been obtained. Twenty of the obtained 500 clones have inhibited the enzyme activity. Three specificity groups were identified for seven of the clones supporting their affinity for different antigenic determinants.     

Affinity modification of DNA-dependent RNA-polymerase from phage T7 with 5'-p-fluorosulfonylbenzoyl adenosine: the effect of modification on the interaction with substrates

T7 RNA polymerase, covalently modified with 5'-p-fluorosulfonylbenzoyl adenosine, looses the ability of binding the promoter (pGEM-2 plasmid) and poly(dC) template as well as the initiating nucleoside triphosphate (GTP). However the enzyme retains the unspecific binding with DNA fragments of considerable length.     

Replication of RNA by the DNA-dependent RNA polymerase of phage T7

The DNA-dependent RNA polymerase of bacteriophage T7 utilizes a specific RNA as a template and replicates it efficiently and accurately. The RNA product (X RNA), approximately 70 nucleotides long, is initiated with either pppC or pppG and contains an AU-tich sequence. Replication of X RNA involves synthesis of complementary strands. Both strands are also significantly self-complementary, producing RNA with an extensive hairpin secondary structure. Replication of X RNA by T7 RNA polymerase is both template and enzyme specific. No other RNA serves as template for replication; neither do other polymerases, including the closely related T3 RNA polymerase, replicate X RNA. The T7 RNA polymerase-X RNA system provides an interesting model for studying replication of RNA by DNA-dependent RNA polymerases. Such a mechanism has been proposed to propagate viroids and hepatitis delta, pathogenic RNAs whose replication seems to depend on cellular RNA polymerases.     

Localization of binding sites of E. coli DNA-dependent RNA-polymerase with photosensitive template analogs

The photoinduced covalent binding of E. coli RNA polymerase with decathymidylic templates containing 5-bromouracil residue has been carried out. Peptides from beta and beta' subunits of the core-enzyme, situated in the DNA-template binding site of the RNA polymerase active center have been localized.     

The role of sulfhydryl groups in the functioning of DNA-dependent RNA-polymerase

Sulfhydryl groups of Escherichia coli DNA-dependent RNA polymerase were chemically modified with alkylating and mercuric-containing compounds. Iodoacetic acid and iodoacetamide were shown not to affect the enzymatic activity, whereas N-ethylmaleimide and mercuric-containing compounds completely inhibit the RNA synthesis. RNA polymerase modified with mercuric ions looses the ability of binding with promoter--containing DNA fragments. Moreover, mercuric ions inhibit the RNA elongation stage. Suggestion is made the Cys residues of RNA polymerase play a key role in double-stranded DNA unwinding. It is shown that SH-groups of beta- and beta'-subunits participate in the binding with double-stranded fragments of DNA.     

The influence of diphosphonic analogues of inorganic pyrophosphate on reactions catalyzed by DNA-dependent RNA-polymerase II

Diphosphonic analogues of inorganic pyrophosphate were studied for their influence both on RNA pyrophosphorolysis and pyrophosphate exchange, catalyzed by purified DNA-dependent RNA-polymerase II from calf thymus. Methylene-, oxyethylene-and aminomethylenediphosphonic acids are shown to compete with PPi for incorporation into nucleoside triphosphate. They activate RNA pyrophosphorolysis in the concentration of 2 mM, but to a less extent than PPi does.     

Development of HIV-1 protease expression methods using the T7 phage promoter system

New and simple human immunodeficiency virus type 1 (HIV-1) protease expression methods in Escherichia coli were developed using the T7 phage promoter system. In order to suppress leaky HIV-1 protease expression under the control of the T7 polymerase, two new methods were tested. One involved the introduction of supplementary T7 promoter regions into host cells [E. coli BL-21(DE3)] containing the HIV-1 protease gene under the control of the T7 promoter. It was expected that the supplementary T7 promoter regions would compete with the HIV-1 protease expression vector for the T7 polymerase binding. The other involved the infection of late-log-phase cultures of E.␣coli JM109 harboring the same HIV-1 protease expression vector with the M13 phage expressing T7 polymerase. Both methods were effective, and transformants with the mature HIV-1 protease expression vector showed ten times higher HIV-1 protease activity than activities obtained with the autoprocessing vector. The expression systems described here are convenient and are also easily applicable for the expression of other proteins toxic for E. coli. Received: 5 September 1996 / Received last revision: 1 November 1996 / Accepted: 15 November 1996     

Localization of low-melting regions in phage T7 DNA

Specific fragmentation of T7 DNA at glyoxal-fixed denatured regions by the S1 endonuclease followed by restriction analysis made it possible to localize four low-melting regions in phage T7 DNA. These regions have the following coordinates:0.5-1.2;14.8+/-0.3;46.3+/-0.5; 98.4+/-0.3 (in T7 DNA length units). The location of the low-melting regions was refined by means of electron-microscopic denaturation mapping and gel electrophoresis of partially denatured DNA. The obtained localization of the low-melting regions is consistent with the available data on the sequence of T7 DNA. The map of low-melting regions was compared with the genetic map of T7 DNA.Images