Characteristics of electrostatic interaction of Escherichia coli RNA polymerase with promoters of T4 phage DNA]

A comparative analysis of electrostatic potential distribution for "early" T4 phage promoters was undertaken. The data obtained indicate that there are some particular elements in the patterns of electrostatic potential distribution of promoter DNA specific for promoter groups differing by their functional response to ADP-ribosylation of the alpha-subunit as well as to rpoB403- or rpoB409 mutationals of the beta-subunit of RNA-polymerase.     

Initiation of RNA molecules by purified Escherichia coli RNA polymerase

Summary The kinesties of appearance of, and the distribution among, the four bases of the chain-initial nucleotides (5-termini) of RNA chains synthesized in vitro under various conditions have been investigated. The results of this study have shown that when native T4 bacteriophage DNA is the template for the RNA polymerase, most chains start with a purine nucleotide. The ratio of ATP termini to GTP termini is independent of the reaction time and of the template/enzyme ratio in the reaction mixture. A similar preferential purine initiation was observed when denatured T4 is the template, but the ratio of ATP to GTP termini is reduced. All 5-termini of poly-AU chains synthesized on poly-dAT templates are ATP.The determination of the kinetics of initiation of RNA chains has allowed direct confirmation of some conclusions which had been inferred previously from sedimentation analyses of the RNA product. (1) Most RNA chains are initiated during a short period at the outset of the reaction. (2) Low concentrations of native DNA templates limit the number of RNA chains synthesized, not the rate of RNA chain growth. (3) The maximum number of RNA molecules which can be synthesized on denatured DNA templates is severalfold larger than the maximum number which can be synthesized on the same weight of native DNA.     

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.     

RNA polymerase of a rifampicin-resistant mutant of Escherichia coli has an altered selectivity to phage T7 DNA promoters

The formation of complexes of RNA polymerases from E. coli W12 and its rpoB409 rifampicin resistant mutant with A1 and D promoters of T7 delta D111 DNA was studied by an abortive RNA synthesis technique. The mutation was shown to affect RNA synthesis initiation at these two promotors differentially so that the efficiency of D promotor utilization is enhanced but the use of A1 promotor is unchanged. The mutation does not interfere with the affinity of the enzyme for both initiating substrates. The results show that the change in RNA polymerase beta-subunit structure has a differential effect on the enzyme interaction with different promotors. The necessity of a classificatory approach to structure-functional analysis of promotors was proposed.