Transcript cleavage by Thermus thermophilus RNA polymerase: Effects of GreA and anti-GreA factors

To gain insight into the mechanism of transcription elongation, the regulation of transcription, and the accessory proteins associated with elongation, we developed a novel procedure for the purification of RNAP from T. thermophilus cells (Xue, Hogan et al. 2000). We characterized the purified enzyme using DNA containing the λP_R promoter (DE13). We demonstrated that T. thermophilus RNAP is capable of binding the λP_R promoter and forming an open complex at 55°C, but incapable of forming a stable open promoter complex and initiating transcription at 37°C. Our results suggest that ambient temperature facilitates the conformational changes associated with specific nucleotide incorporation during elongation.; To investigate the conformational changes that govern transcript cleavage, we examined the effects of neutral salts on the intrinsic transcript cleavage activity of T. thermophilus RNAP (Hogan, Hartsch et al. 2002). Our results indicate that the conformational transitions that control transcript cleavage, and therefore backtracking, are anion dependent.; We have identified a homolog of the prokaryotic transcript cleavage factor GreA in the extreme thermophiles, T. thermophilus and T. aquaticus. The thermostable homologs of GreA share strong sequence identity with other GreA factors, particularly in the N-terminal “basic patch” region. The thermostable GreA factors crosslink to the 3 ′-end of RNA and stimulate the intrinsic cleavage activity of T. thermophilus RNAP. In addition, we have identified a novel transcription factor (Gfh1) in T. thermophilus and T. aquaticus that shares sequence similarity with GreA, but does not function as a transcript cleavage factor. In vitro, Gfh1 functions as an anti-cleavage factor, and does not crosslink to the 3′ -end of RNA. Our data suggest GreA and Gfh1 bind to overlapping sites on RNAP.; Examination of a series of GreA-Gfh1 chimeric proteins reveals that regions within the N-terminal domain of GreA and Gfh1 are responsible for their opposite functions. Our results suggest that “basic patch” residues play a role in the ability of GreA and Gfh1 to contact RNA and either stimulate or inhibit transcript cleavage, but additional residues within the N-terminal domain contribute to the overall function of these two transcription factors.