Inter-subunit recognition and manifestation of segmental mobility in Escherichia coli RNA polymerase: a case study with ω-β' interaction

Abstract

Omega (ω ), 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 ω in which it helps to maintain β' in a correct conformation and recruit it to the α₂β subassembly to form a functional core enzyme (α₂β β'ω). The X-ray structure analysis of Thermus aquaticus core RNA polymerase suggests that two regions of ω latch onto the N-terminal and C-terminal ends of the β'-subunit. In the present study we have monitored the conformational changes in β' as the denatured protein is refolded in the presence and absence of ω using tryptophan fluorescence emission of β' as well as acrylamide quenching of Trp fluorescence. Results indicate that the presence of stoichiometric amounts of ω is helpful in β' refolding. We have also monitored the behavior of the C-terminal tail of ω by engineering three cysteine residues at three different sites in ω and subsequently labeling them with a sulphydryl-specific fluorescent probe. Fluorescence anisotropy measurements of the labeled protein indicate that the C-terminal domain of ω is mobile in the free protein and gets restrained in the presence of β '. Calculations on side-chain interactions show that out of the three mutated positions, two have near neighbourhood interactions only with side-chains in the β' subunit whereas the end of the C-terminal of ω, although it is restrained in the presence of β', has no interacting partner within a 4-Å radius.