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Model of the Mediator middle module based on protein cross-linking.

Larivière L, Plaschka C, Seizl M, Petrotchenko EV, Wenzeck L, Borchers CH, Cramer P - Nucleic Acids Res. (2013)

Bottom Line: The model contains a central tetramer formed by the heterodimers Med4/Med9 and Med7/Med21.The Med7/Med21 heterodimer is flanked by subunits Med10 and Med31.The model is highly extended, suggests that the middle module is flexible and contributes to a molecular basis for detailed structure-function studies of RNA polymerase II regulation.

View Article: PubMed Central - PubMed

Affiliation: Gene Center Munich and Department of Biochemistry, Center for Integrated Protein Science Munich (CIPSM), Ludwig-Maximilians-Universität München, Feodor-Lynen-Str. 25, 81377 Munich, Germany and Department of Biochemistry and Microbiology, Genome British Columbia Protein Centre, University of Victoria, No. 3101-4464 Markham Street, Vancouver Island Technology Park, Victoria, BC V8Z7X8, Canada.

ABSTRACT
The essential core of the transcription coactivator Mediator consists of two conserved multiprotein modules, the head and middle modules. Whereas the structure of the head module is known, the structure of the middle module is lacking. Here we report a 3D model of a 6-subunit Mediator middle module. The model was obtained by arranging crystal structures and homology models of parts of the module based on lysine-lysine cross-links obtained by mass spectrometric analysis. The model contains a central tetramer formed by the heterodimers Med4/Med9 and Med7/Med21. The Med7/Med21 heterodimer is flanked by subunits Med10 and Med31. The model is highly extended, suggests that the middle module is flexible and contributes to a molecular basis for detailed structure-function studies of RNA polymerase II regulation.

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Close-up views of cross-links mapped on various regions of the model. (A) The Med7C/Med21 heterodimer structure. (B) Med4/Med9 heterodimer model. (C) Med4/Med9–Med7C/Med21 tetramer model interface. Subunits are shown as ribbon and colored as in Figure 1. The Cα atoms of cross-linked residues are shown as spheres and labeled. Colored dashed lines indicate the first 10 residues extending from both termini of the model. Black dashed lines indicate inter-subunit or intra-subunit cross-links. A star indicates the intra-subunit crosslink in Med21, which did not fall within the acceptable distance cutoff.
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gkt704-F4: Close-up views of cross-links mapped on various regions of the model. (A) The Med7C/Med21 heterodimer structure. (B) Med4/Med9 heterodimer model. (C) Med4/Med9–Med7C/Med21 tetramer model interface. Subunits are shown as ribbon and colored as in Figure 1. The Cα atoms of cross-linked residues are shown as spheres and labeled. Colored dashed lines indicate the first 10 residues extending from both termini of the model. Black dashed lines indicate inter-subunit or intra-subunit cross-links. A star indicates the intra-subunit crosslink in Med21, which did not fall within the acceptable distance cutoff.

Mentions: The 40 cross-links that we obtained covered all proteins of the middle module except Med31 (Figure 2). Eight cross-link pairs could be mapped on the two known crystal structures of middle module subcomplexes, seven on the Med7C/Med21 subcomplex and one on the Med7N/Med31 subcomplex (Figures 2, 3 and 4A). The mapped cross-link pairs fell within the acceptable distance between Cα atoms of ≤26 Å, which corresponds to the length of the CBDPS spacer (14 Å) plus two times the length of a lysine side chain (6 Å). Only one intra-subunit cross-link in Med21 did not fall within the acceptable distance, possibly because of the flexibility of the protein. This analysis provided a positive control and demonstrated the reliability of our approach. We also performed cross-linking with a preparation of the complete 7-subunit middle module that additionally contained Med1. Owing to the worse biochemical behavior of the sample, however, fewer cross-links were obtained, and no additional cross-link pairs could be identified. We concluded that Med1 does not introduce major structural changes in the middle module, and continued our analysis with the 6-subunit module.Figure 3.


Model of the Mediator middle module based on protein cross-linking.

Larivière L, Plaschka C, Seizl M, Petrotchenko EV, Wenzeck L, Borchers CH, Cramer P - Nucleic Acids Res. (2013)

Close-up views of cross-links mapped on various regions of the model. (A) The Med7C/Med21 heterodimer structure. (B) Med4/Med9 heterodimer model. (C) Med4/Med9–Med7C/Med21 tetramer model interface. Subunits are shown as ribbon and colored as in Figure 1. The Cα atoms of cross-linked residues are shown as spheres and labeled. Colored dashed lines indicate the first 10 residues extending from both termini of the model. Black dashed lines indicate inter-subunit or intra-subunit cross-links. A star indicates the intra-subunit crosslink in Med21, which did not fall within the acceptable distance cutoff.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3814369&req=5

gkt704-F4: Close-up views of cross-links mapped on various regions of the model. (A) The Med7C/Med21 heterodimer structure. (B) Med4/Med9 heterodimer model. (C) Med4/Med9–Med7C/Med21 tetramer model interface. Subunits are shown as ribbon and colored as in Figure 1. The Cα atoms of cross-linked residues are shown as spheres and labeled. Colored dashed lines indicate the first 10 residues extending from both termini of the model. Black dashed lines indicate inter-subunit or intra-subunit cross-links. A star indicates the intra-subunit crosslink in Med21, which did not fall within the acceptable distance cutoff.
Mentions: The 40 cross-links that we obtained covered all proteins of the middle module except Med31 (Figure 2). Eight cross-link pairs could be mapped on the two known crystal structures of middle module subcomplexes, seven on the Med7C/Med21 subcomplex and one on the Med7N/Med31 subcomplex (Figures 2, 3 and 4A). The mapped cross-link pairs fell within the acceptable distance between Cα atoms of ≤26 Å, which corresponds to the length of the CBDPS spacer (14 Å) plus two times the length of a lysine side chain (6 Å). Only one intra-subunit cross-link in Med21 did not fall within the acceptable distance, possibly because of the flexibility of the protein. This analysis provided a positive control and demonstrated the reliability of our approach. We also performed cross-linking with a preparation of the complete 7-subunit middle module that additionally contained Med1. Owing to the worse biochemical behavior of the sample, however, fewer cross-links were obtained, and no additional cross-link pairs could be identified. We concluded that Med1 does not introduce major structural changes in the middle module, and continued our analysis with the 6-subunit module.Figure 3.

Bottom Line: The model contains a central tetramer formed by the heterodimers Med4/Med9 and Med7/Med21.The Med7/Med21 heterodimer is flanked by subunits Med10 and Med31.The model is highly extended, suggests that the middle module is flexible and contributes to a molecular basis for detailed structure-function studies of RNA polymerase II regulation.

View Article: PubMed Central - PubMed

Affiliation: Gene Center Munich and Department of Biochemistry, Center for Integrated Protein Science Munich (CIPSM), Ludwig-Maximilians-Universität München, Feodor-Lynen-Str. 25, 81377 Munich, Germany and Department of Biochemistry and Microbiology, Genome British Columbia Protein Centre, University of Victoria, No. 3101-4464 Markham Street, Vancouver Island Technology Park, Victoria, BC V8Z7X8, Canada.

ABSTRACT
The essential core of the transcription coactivator Mediator consists of two conserved multiprotein modules, the head and middle modules. Whereas the structure of the head module is known, the structure of the middle module is lacking. Here we report a 3D model of a 6-subunit Mediator middle module. The model was obtained by arranging crystal structures and homology models of parts of the module based on lysine-lysine cross-links obtained by mass spectrometric analysis. The model contains a central tetramer formed by the heterodimers Med4/Med9 and Med7/Med21. The Med7/Med21 heterodimer is flanked by subunits Med10 and Med31. The model is highly extended, suggests that the middle module is flexible and contributes to a molecular basis for detailed structure-function studies of RNA polymerase II regulation.

Show MeSH
Related in: MedlinePlus