Postrecruitment Function of Yeast Med6 Protein during the Transcriptional Activation by Mediator Complex
Med6 protein (Med6p) is a hallmark component of evolutionarily conserved Mediator complexes, and the genuine role of Med6p in Mediator functions remains elusive. For the functional analysis of Saccharomyces cerevisiae Med6p (scMed6p), we generated a series of scMed6p mutants harboring a small intern...
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Wiley
2018-01-01
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| Series: | Biochemistry Research International |
| Online Access: | http://dx.doi.org/10.1155/2018/6406372 |
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| author | Gwang Sik Kim Young Chul Lee |
| author_facet | Gwang Sik Kim Young Chul Lee |
| author_sort | Gwang Sik Kim |
| collection | DOAJ |
| description | Med6 protein (Med6p) is a hallmark component of evolutionarily conserved Mediator complexes, and the genuine role of Med6p in Mediator functions remains elusive. For the functional analysis of Saccharomyces cerevisiae Med6p (scMed6p), we generated a series of scMed6p mutants harboring a small internal deletion. Genetic analysis of these mutants revealed that three regions (amino acids 33–42 (Δ2), 125–134 (Δ5), and 157–166 (Δ6)) of scMed6p are required for cell viability and are located at highly conserved regions of Med6 homologs. Notably, the Med6p-Δ2 mutant was barely detectable in whole-cell extracts and purified Mediator, suggesting a loss of Mediator association and concurrent rapid degradation. Consistent with this, the recombinant forms of Med6p having these mutations partially (Δ2) restore or fail (Δ5 and Δ6) to restore in vitro transcriptional defects caused by temperature-sensitive med6 mutation. In an artificial recruitment assay, Mediator containing a LexA-fused wild-type Med6p or Med6p-Δ5 was recruited to the lexA operator region with TBP and activated reporter gene expression. However, the recruitment of Mediator containing LexA-Med6p-Δ6 to lexA operator region resulted in neither TBP recruitment nor reporter gene expression. This result demonstrates a pivotal role of Med6p in the postrecruitment function of Mediator, which is essential for transcriptional activation by Mediator. |
| format | Article |
| id | doaj-art-8347eb36d67c411ab53194dc3a9f1525 |
| institution | Kabale University |
| issn | 2090-2247 2090-2255 |
| language | English |
| publishDate | 2018-01-01 |
| publisher | Wiley |
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| series | Biochemistry Research International |
| spelling | doaj-art-8347eb36d67c411ab53194dc3a9f15252025-02-03T06:08:19ZengWileyBiochemistry Research International2090-22472090-22552018-01-01201810.1155/2018/64063726406372Postrecruitment Function of Yeast Med6 Protein during the Transcriptional Activation by Mediator ComplexGwang Sik Kim0Young Chul Lee1School of Biological Science and Technology, Hormone Research Center, Chonnam National University, Gwangju, Republic of KoreaSchool of Biological Science and Technology, Hormone Research Center, Chonnam National University, Gwangju, Republic of KoreaMed6 protein (Med6p) is a hallmark component of evolutionarily conserved Mediator complexes, and the genuine role of Med6p in Mediator functions remains elusive. For the functional analysis of Saccharomyces cerevisiae Med6p (scMed6p), we generated a series of scMed6p mutants harboring a small internal deletion. Genetic analysis of these mutants revealed that three regions (amino acids 33–42 (Δ2), 125–134 (Δ5), and 157–166 (Δ6)) of scMed6p are required for cell viability and are located at highly conserved regions of Med6 homologs. Notably, the Med6p-Δ2 mutant was barely detectable in whole-cell extracts and purified Mediator, suggesting a loss of Mediator association and concurrent rapid degradation. Consistent with this, the recombinant forms of Med6p having these mutations partially (Δ2) restore or fail (Δ5 and Δ6) to restore in vitro transcriptional defects caused by temperature-sensitive med6 mutation. In an artificial recruitment assay, Mediator containing a LexA-fused wild-type Med6p or Med6p-Δ5 was recruited to the lexA operator region with TBP and activated reporter gene expression. However, the recruitment of Mediator containing LexA-Med6p-Δ6 to lexA operator region resulted in neither TBP recruitment nor reporter gene expression. This result demonstrates a pivotal role of Med6p in the postrecruitment function of Mediator, which is essential for transcriptional activation by Mediator.http://dx.doi.org/10.1155/2018/6406372 |
| spellingShingle | Gwang Sik Kim Young Chul Lee Postrecruitment Function of Yeast Med6 Protein during the Transcriptional Activation by Mediator Complex Biochemistry Research International |
| title | Postrecruitment Function of Yeast Med6 Protein during the Transcriptional Activation by Mediator Complex |
| title_full | Postrecruitment Function of Yeast Med6 Protein during the Transcriptional Activation by Mediator Complex |
| title_fullStr | Postrecruitment Function of Yeast Med6 Protein during the Transcriptional Activation by Mediator Complex |
| title_full_unstemmed | Postrecruitment Function of Yeast Med6 Protein during the Transcriptional Activation by Mediator Complex |
| title_short | Postrecruitment Function of Yeast Med6 Protein during the Transcriptional Activation by Mediator Complex |
| title_sort | postrecruitment function of yeast med6 protein during the transcriptional activation by mediator complex |
| url | http://dx.doi.org/10.1155/2018/6406372 |
| work_keys_str_mv | AT gwangsikkim postrecruitmentfunctionofyeastmed6proteinduringthetranscriptionalactivationbymediatorcomplex AT youngchullee postrecruitmentfunctionofyeastmed6proteinduringthetranscriptionalactivationbymediatorcomplex |