Regulation of transcription through the secondary channel of RNA polymerase

Abstract

The bacterial transcription factor Gre performs the highly conserved function of stimulating the endonucleolytic activity of RNA polymerase for efficient RNA cleavage, thereby promoting transcription elongation and assisting in transcript fidelity. This dynamic factormediated cleavage has been extensively studied, except for within the unusual Cyanobacteria where, notably, no Gre homologues have yet been identified. To investigate this apparent absence of Gre factor, the RNA polymerase of two cyanobacterial species, Synechococcus elongatus 7942 and Synechocystis sp 6803, were purified and tested for their in vitro transcription activity. Rates of intrinsic RNA cleavage were 20-90x greater for the cyanobacterial polymerases than rates found for Escherichia coli. Further study revealed differences in the bridge helix and trigger loop structural elements of the active site as the possible cause of this increased activity. Mutational analyses indicate a reduced flexibility of these elements which may fix the active site into a closed and more hydrolytically competent state. We propose that in cyanobacteria, the lack of Gre factor is compensated by the unique composition and endogenous ability of the polymerase itself to perform fast and efficient transcript cleavage thus eliminating the need for additional factors. In this work a Gre factor homologue, Gfh1, of Thermus aquaticus is also examined. Gfh1 is known to stimulate transcriptional pausing at a wide variety of pause signals and we present further evidence of preferential activity towards the inhibition of transcription from a pre-translocated state