Alternative sigma factors in the free state are equilibrium mixtures of open and compact conformations.

Paromita Raha, Suranjana Chattopadhyay, Srijata Mukherjee, Ruchira Chattopadhyay, Koushik Roy, Siddhartha Roy

Index: Biochemistry 49 , 9809-9819, (2010)

Full Text: HTML

Abstract

Conformational switching upon core RNA polymerase binding is an integral part of functioning of bacterial sigma factors. Here, we have studied dynamical features of two alternative sigma factors. A study of fluorescence resonance energy transfer and hydrodynamic measurements in Escherichia coli σ(32) suggest a compact shape like those found in complex with anti-sigma factors. On the other hand, the fluorescence anisotropy of probes attached to different regions of the protein and previous hydrogen exchange measurements suggest significant internal flexibility, particularly in the C-terminal half and region 1. In a homologous sigma factor, σ(F) of Mycobacterium tuberculosis, emission spectra and fluorescence resonance energy transfer between the single tryptophan (W112) and probes placed in different regions suggest a compact conformation for a major part of the N-terminal half encompassing region 2 and the flexible C-terminal half. Fluorescence anisotropy measurements suggest significant flexibility in the C-terminal half and region 1, as well. Thus, free alternative sigma factors may be in equilibrium between two conformations: a compact one in which the promoter interacting motifs are trapped in the wrong conformation and another less abundant one with a more open and flexible conformation. Such flexibility may be important for promoter recognition and interaction with many partner proteins.