An integrated study of RNAP transcription. (360G-Wellcome-096553_Z_11_Z)

The archaeal RNAP utilises three general transcription factors TBP, TFB and TFE, whichare homologous to TBP, TFIIB and TFIIE in eukaryotes (6,7). TBP binds to the TATAelement of the promoter and recruits TFB that in turn recruits RNAP and forms thepreinitiation complex (PIC). TFE associates with RNAP and stimulates the separation ofpromoter DNA strands via discrete interactions between its two domains (winged helix[WH] and Zinc-ribbon [ZR]) with the RNAP clamp coiled coil and stalk motifs, respectively(Figure 1B). This results in the loading of the template strand into the RNAP activesite(8). In addition, TFE stabilises the PIC by interacting with the nontemplatestrand(8,9). Spt4/5 is universally conserved in evolution(1,10) and, like TFE, it interactswith the RNAP clamp. Spt4/5 can bind to free RNAPs and efficiently inhibits transcriptioninitiation by closing the DNA binding channel of RNAP(10-12) (Figure 1B). In the PIC hisrepression is overcome by TFE, which displaces Spt4/5 from the RNAP. Followinginitiation, TFE can be retained in the transcription elongation complex (TEC, 9), however,Spt4/5 is able to displace TFE by associating with RNAP, which results in an overallstimulation of transcription processivity(8) (Figure 1A/B). Thus, TFE and Spt4/5 competefor the for the binding to RNAP(8) and the binding and displacement plays a pivotal rolefor the progression through the transcription cycle (Figure 1A).Archaeal transcription systems form an interesting mosaic of an RNAPII-like enzyme thatis regulated by both eukaryote- and bacterial-like transcription factors. In bacteria, NusG(Spt5) has been shown to interact with the elongation factor NusE(4), which is identical tothe ribosomal protein S10. The NusG-NusE interaction could provide the long soughtafter physical link between the transcription and translation machineries (4,5). Archaeaare, like bacteria, prokaryotes and their transcription and translation processes arecoupled(13). Considering the evolutionary conservation of NusG/Spt5 and NusE(S10) itis likely that the interaction between them also connects the two processes in archaea.During termination, the inherently stable transcription elongation complex dissociates,which requires an opening of the RNAP clamp(14),(2). The precise molecularmechanisms are unknown but would seem to require the dissociation of Spt4/5 from theelongation complex, and involve pausing and possibly backtracking - a retrogrademovement of RNAP(5,15). A short poly-U stretch is sufficient to trigger termination ofarchaeal RNAP (16-19). This process readily occurs in vitro without exogenous factorssuch as the bacterial rho helicase, however, the archaeal helicase MJ0669 has beenproposed to function as archaeal termination factor in vivo. Rho is chiefly recruited to theTEC via interactions with the KOW domain of NusG; by analogy it is possible thatMJ0669 is recruited to archaeal TEC via the KOW domain of Spt5. Thus, multipleprocesses converge on RNAP-bound Spt4/5 and thereby regulate transcription.Werner, WT New Investigator Award, body text2The molecular mechanisms outlined above are at best partially understood and severalof the components have not been characterised at all. The function of RNAP subunitsRpo8, Rpo13(20,21) and C34(22) is completely unknown. However, based on theirproximity to the DNA binding channel (Rpo13) and NTP pore (Rpo8) of RNAP we havedeveloped good lead hypotheses regarding their roles during transcription initiation,elongation and termination.The Big Open Questions in the field concern the dynamic aspects of transcription. The12-subunit RNAP (~370 kD) has distinct conformations in the initiation and elongationcomplex, and in the free enzyme – but the transitions between the conformational stateshave never been observed in solution and in real time. Transcription factors are thoughtto aid the changes between the initiation into the elongation complex, and duringtranscription termination – but the mechanisms are not understood. Likewisetranscription factors consist of multiple domains that are rearranged during RNAPbinding. We have developed a fluorescence-based system capable of monitoringconformational changes in RNAP(23) and factors, and have shown that both the RNAPstalk, TFE and Spt4/5 modulate the position of the RNAP clamp in ensemble and singlemolecule FRET experiments (manuscript in preparation). During the transcription cycledistinct protein-protein interactions need to be disrupted (e.g. TFB-RNAP, TFE-RNAP)while others need to be established (e.g. Spt4/5-RNAP) in a sequential manner(8,24,25)– and we have the experimental methods to characterise them. Factors including TBPand TFB are recruited to and dissociate from the gene-specific promoters in a dynamicfashion (2,26). In response to environmental changes (e.g. nutrient shifts) or insults (e.g.UV and heat-shock) the distribution of the transcription apparatus is changingconcomitantly with the gene expression pattern, but it is currently not understood howRNAP and transcription factors (and ribosomes) interact to orchestrate this process. Inorder to elucidate all of the above we want to characterise (i) RNAP/factor mechanismsand interactions in vitro, (ii) the distribution of RNAP/factors on a global level in vivo, and(iii) the whole genome expression profiles in archaea under a range of environmentalconditions.