The model of amyloid aggregation of Escherichia coli RNA polymerase σ70 subunit based on AFM data and in vitro assays.

To propose a model for recently described amyloid aggregation of E.coli RNA polymerase σ(70) subunit, we have investigated the role of its N-terminal region. For this purpose, three mutant variants of protein with deletions Δ1-73, Δ1-100 and Δ74-100 were constructed and studied in a series of in vitro assays and using atomic force microscopy (AFM). Specifically, all RNA polymerase holoenzymes, reconstituted with the use of mutant σ subunits, have shown reduced affinity for promoter-containing DNA and reduced activity in run-off transcription experiments (compared to that of WT species), thus substantiating the modern concept on the modulatory role of N-terminus in formation of open complex and transcription initiation. The ability of mutant proteins to form amyloid-like structures has been investigated using AFM, which revealed the increased propensity of mutant proteins to form rodlike aggregates with the effect being more pronounced for the mutant with the deletion Δ1-73 (10 fold increase). σ(70) subunit aggregation ability has shown complex dependence on the ionic surrounding, which we explain by Debye screening effect and the change of the internal state of the protein. Basing on the obtained data, we propose the model of amyloid fibril formation by σ(70) subunit, implying the involvement of N-terminal region according to the domain swapping mechanism.

Isolation and properties of human transketolase.

Recombinant human (His)(6)-transketolase (hTK) was obtained in preparative amounts by heterologous expression of the gene encoding human transketolase in Escherichia coli cells. The enzyme, isolated in the form of a holoenzyme, was homogeneous by SDS-PAGE; a method for obtaining the apoenzyme was also developed. The amount of active transketolase in the isolated protein preparation was correlated with the content of thiamine diphosphate (ThDP) determined in the same preparation. Induced optical activity, facilitating studies of ThDP binding by the apoenzyme and measurement of the transketolase reaction at each stage, was detected by circular dichroism spectroscopy. A single-substrate reaction was characterized, catalyzed by hTK in the presence of the donor substrate and in the absence of the acceptor substrate. The values of the Michaelis constant were determined for ThDP and a pair of physiological substrates of the enzyme (xylulose 5-phosphate and ribose 5-phosphate).

Purification of core enzyme of Escherichia coli RNA polymerase by affinity chromatography.

A method for isolation of a highly purified preparation of E. coli RNA polymerase core enzyme was developed based on IMPACT technology and dissociation of the RNA polymerase complex with sigma(70) subunit. Washing of the immobilized RNA polymerase with 5-10 mM solution of glutamate (pH 5.0-5.5) completely removed the sigma(70) subunit from the holoenzyme and decreased amounts of protein admixtures. The possibility of reconstruction of the RNA polymerase holoenzyme directly on the affinity column was demonstrated. Activities of the resulting RNAP core enzyme preparations were tested by in vitro transcription. Some amino acids and their mixtures were shown to influence the in vitro transcription. The findings indicate that changes in the transcription efficiency in the presence of amino acids should be associated with a specific destruction of the interaction between sigma(70) subunit and the core enzyme.

A system for heterologous expression and isolation of Escherichia coli RNA polymerase and its components.

A set of plasmid vectors for expression of all major Escherichia coli RNA polymerase subunits as fusion proteins with intein- and chitin-binding domains, allowing protein purification in accordance with IMPACT technology, was constructed. It is demonstrated that the fusion subunits alpha, beta or beta' in conjunction with the natural subunits alpha, beta, beta', and sigma can participate in RNA polymerase assembly in vivo, providing affinity-based isolation of the enzyme. Functional activity of the enzyme preparations was demonstrated in the experiments on in vitro transcription and promoter complex formation. With the use of IMPACT technology, sigma(70) subunit can be isolated as an individual protein without admixture of RNA polymerase.