Several mechanisms have been proposed for the pol I transcript termination in Schizosaccharomyces pombe. Two well known models are “Pause and Release” and “Torpedo”. Each mechanism explains the role of some of the cis- and trans-factors in transcript termination and the eventual maturation of the ribosomal RNA, but neither mechanism can explain all the experimental observations. A recent study has suggested that each of the two mechanisms can terminate the pol I transcription independently but with significantly less efficiency than the presence of both mechanisms. To help clarify the reasons for the discrepancies in these data, in this study the suggested mechanisms were examined further in three areas by using alternative techniques. First, the effect of uracil concentration or selection times on the transformation frequency of alternative 3’external transcribed spacer (3’ETS) constructs were assessed. Consistent with the previous results a construct containing the full 3’ETS showed the higher transformation frequencies compared with a construct containing only the hairpin or only the termination sites. However, results showed neither the uracil concentration nor selection times have a significant effect on the transformation frequency. Second, to further confirm the “pause and release” mechanism, the termination sites identified by S1 nuclease studies were analyzed using ligation-mediated RT-PCR. The 3’ terminus of the mature 25S rRNA was demonstrated readily but, unexpectedly, the 3’termini of the 3’ETS precursor molecules were not detected, possibly because of their specific structure. Finally, the 3’ extended rRNA precursors were studied by semi-quantitative RT-PCR. These appeared not to correspond with past nuclease protection analyses nor did they demonstrate downstream exonuclease function, observations which question our current understanding of Pol I transcript termination.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:OGU.10214/7289 |
| Date | 24 July 2013 |
| Creators | Vazin, Mahsa |
| Contributors | Nazar, Ross N |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | Thesis |
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