Seminar

Transcription initiation and elongation factors compete for binding on the RNAP clamp during the transcription cycle

Finn Werner, PhD

Transcription of eukaryotic RNAPII and the closely related archaeal RNAP is facilitated by homologous transcription initiation and elongation factors. TFIIE and the archaeal homologue TFE enhance DNA strand separation during initiation by an unknown mechanism. We have developed a fluorescently labelled recombinant M. jannaschii RNAP system to probe the structure of the archaeal transcription initiation complex, consisting of promoter DNA, TBP, TFB, TFE and RNAP. We have localised the position of the TFE winged helix (WH-) and Zinc-ribbon (ZR-) domains on the RNAP using single molecule FRET, and obtained information about their location relative to the nontemplate strand of the promoter using fluorescence quenching. Based on the positions of the TFE domains and the nontemplate strand (NTS) of the promoter we have developed specific structural hypotheses for the function of TFE during transcription initiation. The binding site of the TFE WH-domain on the RNAP clamp coiled coil overlaps with the binding site of the NGN domain of the universally conserved elongation factor Spt4/5 (NusG). We show that both factors compete for RNAP binding in vitro. Association of Spt4/5 with RNAP represses promoter-directed transcription in the absence of TFE, which alleviates the inhibition by displacing Spt4/5 from RNAP. During the elongation phase of transcription, the roles are reversed, as Spt4/5 can displace TFE from the RNAP elongation complex and stimulate processivity. Despite the close resemblance to RNAPII, the archaeal transcription complex structure and protein-protein interaction network is surprisingly similar to the bacterial system. The universally conserved RNAP clamp coiled coil serves as binding site for bacterial and archaeo-eukaryotic initiation factors, region 2 of sigma 70 and the TFE WH-domain, respectively, and in addition interacts with elongation factors, NusG/RfaH and Spt4/5, respectively. However, whereas the elongation factors are evolutionary related, the initiation factors are not and the similar interaction mode is likely to be the outcome of convergent evolution. This highlights the importance of the RNAP clamp as a versatile target for factors involved in both transcription initiation and elongation.