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The ω subunit of Escherichia coli RNA polymerase, consisting of 90 amino acids, is present in stoichiometric amounts per molecule of core RNA polymerase (α2ββ′). The presence of ω is necessary to restore denatured RNA polymerase in vitro to its fully functional form, and, in an ω-less strain of E. coli, GroEL appears to substitute for ω in the maturation of RNA polymerase. The X-ray structure of Thermus aquaticus core RNA polymerase suggests that two regions of ω latch on to β′ at its N-terminus and C-terminus. We show here that ω binds only the intact β′ subunit and not the β′ N-terminal domain or β′ C-terminal domain, implying that ω binding requires both these regions of β′. We further show that ω can prevent the aggregation of β′ during its renaturation in vitro and that a V8-protease-resistant 52-amino-acid-long N-terminal domain of ω is sufficient for binding and renaturation of β′. CD and functional assays show that this N-terminal fragment retains the structure of native ω and is able to enhance the reconstitution of core RNA polymerase. Reconstitution of core RNA polymerase from its individual subunits proceeds according to the steps α + α → α2 + β → α2β + β′ → α2ββ′. It is shown here that ω participates during the last stage of enzyme assembly when β′ associates with the α2β subassembly. [ABSTRACT FROM AUTHOR]

Examines the involvement of GroEl in the activation of Escherichia coli RNA polymerase. Function of protein; Copurification of the polypeptide with RNA polymerase; Interaction with unstable protein-folding intermediate.

Among various group 1 sigma factors, two amino acids. Va155 and A1a59 are the conserved amino acids in the 1.1 hydrophobic subdomain. These two sites have been mutated to generate variants designated as [Gly55]σ70 and [Gly59)σ70, where glycine replaces valine and alanine, respectively. The function of these sigma mutants is reported here. The molecular mass of these proteins determined on denaturing gels was 70 kDa, which is the expected calculated molecular mass; wild-type σ70 has an apparent molecular mass of 87 kDa. However, [Gly434]σ70 which contains a mutation at the DNA-binding rpoD box region, also migrates as a 70-kDa protein on SDS/PAGE. Circular dichroism spectral analysis indicated that both [Gly55]σ70 and [Gly59]σ70 have reduced helicity (20%) compared to wild-type σ70 (50%). Binding of sigma factors with the hydrophobic, surface active probe I-anilinonapthalene-8-sulphonate, has shown that more hydrophobic suites are available/exposed in [Gly55]σ70. [Gly59]σ70 as well as in [Gly434]σ70 in comparison to wild-type σ70. Time-resolved emission spectroscopic studies have suggested transient binding between these mutants and DNA. The different holoenzyme RNA polymerases generated upon reconstituting these mutants independently with core RNA polymerase (α2ββ') have shown reduced trunscnptional activity in comparison to the enzyme containing wild-type sigma factor. However; another mutation (Val→Gly) in the hydrophobic subdomain 1.2 at position 83, which is designated as [Gly83] σ70, has similar properties as the wild-type with respect to its mobility on denaturing gels, circular dichroism profile, and transcriptional activity when reconstituted with core RNA polymerase. It appears that the 1.1 subdomain in σ70 may interact hydrophobically with the 2.3/2.4 DNA-binding region. [ABSTRACT FROM AUTHOR]