Interaction of bacterial RNA-polymerase with two different promoters of phage T7 DNA. Conformational analysis.

Using a rifampicin-resistant RNA polymerase with altered specificity to different promoters, the D promoter of T7 phage DNA with increased affinity to the mutant enzyme was chosen. This promoter and the T7 A1 promoter with unchanged affinity as well as some nonpromoter DNA fragments were used to compare temperature-induced conformational transitions of RNA polymerase in the course of complex formation. Conformational alterations of RNA polymerase were monitored by the fluorescent label method.

Specific modification of Escherichia coli RNA polymerase with monomercury derivative of fluorescein acetate.

The method of specific modification of RNA polymerase with a monomercuric fluorescein derivative, fluorescein-monomercuriacetate (FMMA), is proposed. Under an appropriate condition of modification, FMMA is capable of mercaptid bonding with one of the alpha-subunits. It is shown that covalent modification with FMMA does not affect the kinetic parameters (KB and k2) of RNA synthesis nor does it lead to the inhibition of the overall RNA synthesis.

[Specific modification of the alpha-subunit of Escherichia coli Rna polymerase by monomercuric derivative of fluorescein mercuric acetate].

The method for specific modification of Escherichia coli RNA polymerase by a monomercuric derivative of fluorescein--fluoresceinmonomercuracetate (FMMA)--a specific reagent for SH-groups of proteins is suggested. It is shown, that in conditions of equimolar FMMA/enzyme ratio the fluorescent label interacts preferantially with a single sulfhydryl group in alpha-subunit of RNA polymerase. The mercaptide bonding formation is followed by significant alterations of all spectral parameters of FMMA, but has no effect on the kinetic parameters (KB and k2) of RNA synthesis initiation nor does it lead to inhibition of the total RNA synthesis.

[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.