Abstract
Transcription is the most fundamental step in gene expression in any living organism. Various environmental cues help in the maturation of core RNA polymerase (RNAP; α(2)ββ'ω) with different σ-factors, leading to the directed recruitment of RNAP to different promoter DNA sequences. Thus it is essential to determine the σ-factors that affect the preferential partitioning of core RNAP among various σ-actors, and the role of σ-switching in transcriptional gene regulation. Further, the macromolecular assembly of holo RNAP takes place in an extremely crowded environment within a cell, and thus far the kinetics and thermodynamics of this molecular recognition process have not been well addressed. In this study we used a site-directed bioaffinity immobilization method to evaluate the relative binding affinities of three different Escherichia coli σ-factors to the same core RNAP with variations in temperature and ionic strength while emulating the crowded cellular milieu. Our data indicate that the interaction of core RNAP-σ is susceptible to changes in external stimuli such as osmolytic and thermal stress, and the degree of susceptibility varies among different σ-factors. This allows for a reversible σ-switching from housekeeping factors to alternate σ-factors when the organism senses a change in its physiological conditions.