DISTINCT FUNCTIONS OF N-TERMINAL AND C-TERMINAL DOMAINS OF GREA, AN ESCHERICHIA-COLI TRANSCRIPT CLEAVAGE FACTOR

The prokaryotic transcription factors GreA and GreB are involved in th e regulation of transcript elongation by RNA polymerase (RNAP). Their known activities include suppression of transcription arrest, enhancem ent of transcription fidelity, and facilitation of the transition from abortive initiation to productive elongation. Presumably, Gre protein s exert their functions by altering the conformation of the enzyme in ternary elongation complexes (TEC) and inducing the cleavage of nascen t RNA. GreA and GreB have a similar structural organization and consis t of two domains: a C-terminal globular and an extended N-terminal coi led-coil domain. To investigate the functional roles of Gre domains, w e expressed separately the N and C-terminal domains of GreA (NTD and C TD, respectively) and characterized their activities with in vitro ass ays. We demonstrate that the NTD possesses the residual transcript cle avage activity of the wild-type GreA. The CTD does not display any nuc leolytic activity; however, it substantially increases the cleavage ac tivity of the NTD. In contrast to NTD, the CTD competes with GreA and GreB for binding to RNAP and inhibits their transcript cleavage and an tiarrest activities. Both domains individually and together inhibit tr anscription elongation. From these results we conclude that the NTD is responsible for the GreA induction of nucleolytic activity while the CTD determines the binding of GreA to RNAP. Both domains are required for full functional activity of GreA. (C) 1998 Academic Press Limited.