Termination of Ribosomal RNA Transcription by RNA Polymerase I in Schizosaccharomyces pombe

Termination of ribosomal RNA transcripts in Schizosaccharomyces pombe is a crucial process that occurs downstream of the mature 25S rDNA sequence. Two different models have been proposed for this termination process, the “Pause and release” and the “Torpedo” models. Previous SI nuclease mapping studies have indicated that termination occurs at three different sites (T1, T2 and T3) +267, + 338 and + 448 respectively downstream of the 25S rRNA. It is believed that 90% of the termination occurs at the first termination site (T1). In this thesis, the relative contribution and the termination efficiency of the two proposed mechanisms were examined further using S. pombe cells transformed with different mutations. An alternate technique, RT-PCR, was used to remap the 3’ ETS region leading to better understanding of the two mechanisms and enzymes that underlie them. The study also investigated the effect of exonuclease activity on processed fragments generated by PacI cleavage. The results showed that both the “Torpedo” and the “Pause and release” mechanisms can terminate Pol I transcription efficiently. The study also showed that exonuclease degrades processed 3’ ETS fragments after their release by PacI cleavage. Inhibition of PacI cleavage by either RNA sequence deletion or protein mutation resulted in transcripts terminating fully at the first termination site with no transcripts detected beyond T1. On the other hand, deletion of the cognate termination elements resulted in read-through transcription after the rDNA region. The research is consistent with the presence of two redundant termination mechanisms, which provide fail-safe termination to ensure that the downstream sequence is not disrupted by read-through transcription.