Abstract
The DNA damage response is an essential process for the survival of living cells. In a subset of stress-responsive genes in humans, Elongin controls transcription in response to multiple stimuli, such as DNA damage, oxidative stress, and heat shock. Yeast Elongin (Ela1-Elc1), along with Def1, is known to facilitate ubiquitylation and degradation of RNA polymerase II (pol II) in response to multiple stimuli, yet transcription activity has not been examined. We have found that Def1 copurifies from yeast whole-cell extract with TFIIH, the largest general transcription factor required for transcription initiation and nucleotide excision repair. The addition of recombinant Def1 and Ela1-Elc1 enhanced transcription initiation in an in vitro reconstituted system including pol II, the general transcription factors, and TFIIS. Def1 also enhanced transcription restart from TFIIS-induced cleavage in a pol II transcribing complex. In the Δdef1 strain, heat shock genes were misregulated, indicating that Def1 is required for induction of some stress-responsive genes in yeast. Taken together, our results extend the understanding of the molecular mechanism of transcription regulation on cellular stress and reveal functional similarities to the mammalian system.
| Original language | English |
|---|---|
| Pages (from-to) | 13230-13235 |
| Number of pages | 6 |
| Journal | Proceedings of the National Academy of Sciences of the United States of America |
| Volume | 114 |
| Issue number | 50 |
| DOIs | |
| State | Published - 12 Dec 2017 |
Bibliographical note
Funding Information:We thank Craig Kaplan and Yunye Zhu (Texas A&M University) for their help with analysis of HSP12 expression; Hua-Ying Fan, Hillary Nelson, and Rina Fujiwara (University of Pennsylvania) and P.-J. Mattei and Ralph Davis (Stanford University) for critical reading of the manuscript and helpful discussions; Dong Wang (University of California, San Diego) for providing recombinant Rad26; and Jeremy Wilusz, Chie Arai, and Deirdre Tatomer (University of Pennsylvania) for technical support in qPCR and Western blot analyses. The N-terminal Myc-tagged Def1 yeast strain was a gift from the Svejstrup laboratory at the Francis Crick Institute. This research was supported by National Institutes of Health (NIH) Grant GM123233. J.Z. and N.K. are funded by Israel Science Foundation Grant 15/1768. T.V.E. is supported by Structural Biology and Molecular Biophysics Training Program Grant GM008275. E.L.-S. is funded by NIH Grant 1 F31 GM123744-01.
Funding Information:
ACKNOWLEDGMENTS. We thank Craig Kaplan and Yunye Zhu (Texas A&M University) for their help with analysis of HSP12 expression; Hua-Ying Fan, Hillary Nelson, and Rina Fujiwara (University of Pennsylvania) and P.-J. Mattei and Ralph Davis (Stanford University) for critical reading of the manuscript and helpful discussions; Dong Wang (University of California, San Diego) for providing recombinant Rad26; and Jeremy Wilusz, Chie Arai, and Deirdre Tatomer (University of Pennsylvania) for technical support in qPCR and Western blot analyses. The N-terminal Myc-tagged Def1 yeast strain was a gift from the Svejstrup laboratory at the Francis Crick Institute. This research was supported by National Institutes of Health (NIH) Grant GM123233. J.Z. and N.K. are funded by Israel Science Foundation Grant 15/1768. T.V.E. is supported by Structural Biology and Molecular Biophysics Training Program Grant GM008275. E.L.-S. is funded by NIH Grant 1 F31 GM123744-01.
Keywords
- Def1
- RNA polymerase II
- Stress response
- TFIIH
- Transcription