Inter-subunit recognition and manifestation of segmental mobility in Escherichia coli RNA polymerase: a case study with $\omega-\beta′$ interaction

Abstract

Omega $(\omega)$, consisting of 91 amino acids, is the smallest of all the Escherichia coli RNA polymerase subunits and is organized into an N-terminal domain of 53 amino acids followed by an unstructured tail in the C-terminal region. Our earlier experiments have shown a chaperone-like function of \omega in which it helps to maintain \beta' in a correct conformation and recruit it to the $\alpha_2 \beta$ subassembly to form a functional core enzyme $(\alpha_2 \beta \beta'\omega)$. The X-ray structure analysis of Thermus aquaticus core RNA polymerase suggests that two regions of \omega latch onto the N-terminal and C-terminal ends of the \beta'-subunit. In the present study we have monitored the conformational changes in \beta' as the denatured protein is refolded in the presence and absence of \omega using tryptophan fluorescence emission of \beta' as well as acrylamide quenching of Trp fluorescence. Results indicate that the presence of stoichiometric amounts of \omega is helpful in \beta' refolding. We have also monitored the behavior of the C-terminal tail of \omega by engineering three cysteine residues at three different sites in \omega and subsequently labeling them with a sulphydryl-specific fluorescent probe. Fluorescence anisotropy measurements of the labeled protein indicate that the C-terminal domain of \omega is mobile in the free protein and gets restrained in the presence of \beta'. Calculations on side-chain interactions show that out of the three mutated positions, two have near neighbourhood interactions only with side-chains in the \beta' subunit whereas the end of the C-terminal of \omega, although it is restrained in the presence of \beta', has no interacting partner within a 4-Å radius