Comparative studies on elongation and termination of the three RNA polymerases from S. cerevisiae in vitro

In Saccharomyces cerevisiae (hereafter called yeast), three RNA polymerases synthesize the nuclear transcriptome. During the transcriptional cycle, RNA polymerase I, II and III (Pol I, II and III) are recruited to their respective promoter, elongate the nascent transcript in the context of chromatin and finally terminate transcription. Whereas the initation step is quite well understood, our knowledge about elongation and termination is very limited. Therefore, we developed an in vitro transcription system in which elongation and termination of RNA Pol I can be analyzed independent of promoter-specific initiation. The same transcription system allowed comparison of the three RNA polymerases regarding their behaviour, if barriers are encountered during elongation. In summary, the following questions were addressed: 1) Several DNA cis elements and trans-acting factors are described to support RNA Pol I dependent transcription termination. Among the protein factors are the Reb1 homolog Ydr026c/Nsi1 which was recently found to be required for efficient Pol I termination in vivo, Reb1, which terminates transcription in vitro and the replication fork blocking protein Fob1. We further investigated the role of these factors in the termination process regarding i) the role of Nsi1 and other possible trans-acting factors and ii) the influence of mutations in Pol I subunits in transcription termination. We found that Nsi1 imposed an elongation barrier for Pol I. Furthermore, we could show that presence of the 35S rDNA terminator-proximal Reb1 binding site was sufficient for Nsi1-dependent Pol I pausing/termination in vitro which was further enhanced by the T-rich 1 element. In addition, our data suggest that Nsi1 and Fob1 exhibit a cooperative effect in Pol I transcription termination in vitro. 2) We further wanted to elucidate whether RNA Pol I, II and III deal differently with elongation obstacles in general. Thus, we comparatively analyzed Pol I, II and III transcription of templates complexed with the physiological elongation barriers Nsi1, Reb1 and Fob1 in vitro. Furthermore, the unphysiological bacterial strong DNA-binding proteins LexA and LacI as well as the mouse rDNA terminaton factor TTF-I were included in the analysis. We could show that Nsi1 and Reb1 were specifically impairing Pol I transcription elongation. Contrary, LexA only imposed a road-block for Pol II. On the other hand, TTF-I was an elongation obstacle for all three RNA polymerases. 3) In all eukaryotic cells, gene transcription occurs in the context of chromatin. Chromatin is a complex assembly of DNA and DNA bound proteins, with histone molecules forming the abundant, basic unit of chromatin, the nucleosome. It is of great importance to understand how the different RNA polymerases cope with the chromatin template. It has been suggested, that actively transcribed Pol I and III genes are devoid of nucleosomes, whereas Pol II genes are nucleosomal. To analyze whether the RNA polymerases have intrinsic activities to deal differently with the nucleosomal templates, we comparatively investigated Pol I, II and III transcription elongation on templates complexed with nucleosomes in vitro. Many studies employing in vitro assays have already analyzed chromatin transcription by the different RNA Pols. However, it is hard to compare the individual results, since the respective in vitro systems used for analysis often differ significantly in many aspects, whereas side-by-side comparison of Pol I, II and III is possible in our system. Furthermore, we used RNA Pol I mutants to elucidate the contribution of certain subunits to transcribe through nucleosomal templates. We observed that Pol I and III were able to transcribe a nucleosomal template in vitro, whereas in agreement with the literature a single nucleosome imposed a very strong barrier for Pol II elongation. Additionally, we could demonstrate that subunit A49 significantly contributes to Pol I’s ability to overcome the nucleosomal barrier.