DOMAIN ORGANIZATION OF ESCHERICHIA-COLI TRANSCRIPT CLEAVAGE FACTORS GREA AND GREB

The GreA and GreB proteins of Escherichia coli induce cleavage of the nascent transcript in ternary elongation complexes of RNA polymerase. Ore factors are presumed to have two biologically important and evolut ionarily conserved functions: the suppression of elongation arrest and the enhancement of transcription fidelity. A three-dimensional struct ure of GreB was generated by homology modeling on the basis of the kno wn crystal structure of GreA. Both factors display similar overall arc hitecture and surface charge distribution, with characteristic C-termi nal globular and N-terminal coiled-coil domains. One major difference between the two factors is the ''basic patch'' on the surface of the c oiled-coil domain, which is much larger in GreB than in GreA. In both proteins, a site near the basic patch cross-links to the 3' terminus o f RNA in the ternary transcription complex. GreA/GreB hybrid molecules were constructed by genetic engineering in which the N-terminal domai n of one protein was fused to the C-terminal domain of the other. In t he hybrid molecules, both the coiled-coil and the globular domains con tribute to specific binding of Gre factors to RNA polymerase, whereas the antiarrest activity and the GreA or GreB specificity of transcript cleavage is determined by the N-terminal domain. These results implic ate the basic patch of the N-terminal coiled-coil domain as an importa nt functional element responsible for the interactions with nascent tr anscript and determining the size of the RNA fragment to be excised du ring the course of the cleavage reaction.