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Karakasili, Eleni (2010): Molecular mechanism for degradation of transcriptionally stalled RNA polymerase II in the yeast Saccharomyces cerevisiae.. Dissertation, LMU München: Faculty of Chemistry and Pharmacy
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Abstract

Transcription of protein coding genes by RNA polymerase II (RNAPII) is an essential step in gene expression. Transcription elongation is a highly dynamic and discontinuous process that includes frequent pausing of RNAPII, backtracking, and arrest both in vitro and in vivo. Consequently, a multitude of transcription elongation factors are needed for efficient transcription elongation. When transcription elongation factors fail to “restart” RNAPII the persistently stalled RNAPII complex prevents transcription and thus has to be recognized and removed to free the gene for subsequent polymerases. Similarly, DNA damage causes stalling of RNAPII. In this case, the DNA damage is either repaired by Transcription-Coupled Repair (TCR) or RNAPII is degraded as a “last resort” mechanism by the ubiquitin proteasome system. In contrast to RNAPII degradation caused by DNA damage, the cellular pathway for removal of transcriptionally stalled RNAPII complexes has remained largely obscure. However, it was speculated that transcriptionally stalled RNAPII complexes are degraded by the same pathway as RNAPII stalled due to DNA damage. Here, it is shown that the pathway for degradation of transcriptionally stalled RNAPII is distinct from the DNA damage-dependent pathway, providing the first evidence that the cell distinguishes between RNAPII complexes stalled for different reasons. The novel cellular pathway for transcriptional stalling-dependent degradation of RNAPII is termed TRADE. Specifically, in the TRADE pathway a different yet overlapping set of enzymes is responsible for poly- and de-ubiquitylation of transcriptionally stalled RNAPII. Moreover, the catalytic 20S proteasome is recruited to transcribed genes indicating that Rpb1 of transcriptionally stalled RNAPII complexes is degraded at the site of transcription. Importantly, nucleotide starvation and temperature stress which might mimic natural conditions of transcription elongation impairment also lead to RNAPII degradation. Finally, this study provides the first evidence that the mechanism for the controlled degradation of the transcriptionally stalled RNA polymerase complex might also exist for transcription by RNAPI and RNAPIII. Taken together, the TRADE pathway elucidated in this study ensures continued transcription.