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| - | The bacterial transcription-repair coupling factor TRCF, also called Mfd translocase, is a DNA repair protein. It has a role in transcription-coupled repair, a subpathway of nucleotide excision repair (NER). Mfd recognizes stalled RNA polymerase and either restarts transcription or removes the stalled polymerase and recruits the NER proteins UvrA and UvrB. | + | The bacterial transcription-repair coupling factor TRCF, also called Mfd translocase, is a DNA repair protein. It has a role in transcription-coupled repair, a subpathway of nucleotide excision repair (NER). Mfd recognizes stalled RNA polymerase (RNAP) and either restarts transcription or removes the stalled polymerase and recruits the NER proteins UvrA and UvrB. |
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| | == Function == | | == Function == |
| - | '''Transcription-repair coupling factor (TRCF)''' or '''Mfd''' enables the coupling of these processes in bacteria and humans. The TRCF has ATPase activity. <scene name='2eyq/Asymmunit/2'>The crystallographic asymmetric unit of the solved structure contains 2 monomers of Mfd (residues 2-1147 of 1151 for one molecule and 5-1147 of the other), five HEPES molecules, and the sulfate ions.</scene>
| + | Mfd has ATP hydrolysis activity, DNA binding sites and a UvrA binding sites. These three functions are inhibited in the isolated enzyme, but are activated when Mfd encounters stalled RNA polymerase (or through various mutations that remove inhibitory domains <ref>PMID: 27864884</ref>). Mfd also contains an RNA interaction domain (RID) that binds to the beta subunit of RNAP. |
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| - | Size exclusion chromatogaphy indicates that the protein <scene name='2eyq/Biolunit/1'>exists as a monomer in solution.</scene> | |
| | == Structural highlights == | | == Structural highlights == |
| | <StructureSection load='' size='350' side='right' scene='2eyq/Domainscolorsflabe/3' caption=''> | | <StructureSection load='' size='350' side='right' scene='2eyq/Domainscolorsflabe/3' caption=''> |
| - | <scene name='2eyq/Figonea/2'>The monomer colored as</scene> an N→C rainbow similar to [http://www.sciencedirect.com/science?_ob=MiamiCaptionURL&_method=retrieve&_udi=B6WSN-4J79KGF-J&_image=fig1&_ba=1&_user=10&_rdoc=1&_fmt=full&_orig=search&_cdi=7051&view=c&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=a4326e4023d32f5de977da04b9a9fdf6 Figure 1A] of [http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6WSN-4J79KGF-J&_user=10&_rdoc=1&_fmt=&_orig=search&_sort=d&_docanchor=&view=c&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=cd0b2f15037bec97859866e118038962 the article describing] the structure. {{Link Toggle FancyCartoonHighQualityView}}.
| + | The initial scene shows the domains of Mfd in the conformation of the apo-enzyme. The UvrA interaction site (on domain 2) is occluded by domain 7. The translocase domains (domain 5 and domain 6), through interactions with domains 1 and 3, are locked in an inactive conformation, preventing the typical hinge motion of translocases when they bind and hydrolyze ATP while moving along DNA. |
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| - | The domains in the structure of Mfd:
| + | When <scene name='46/460252/Mfd_rnap_l1/1'>Mfd binds to a stalled RNA polymerase</scene>, the interaction of the RID (domain 4) with RNA polymerase, combined with interactions of domains 5 and domain 6 with DNA and binding to ATP lead to conformational changes that disrupt inter-domain interactions seen in the apo-structure and activate the translocase activity of Mfd. The structures of several intermediates in this process were determined using cryo-EM, shedding light on the activation mechanism, and how translocation of Mfd on DNA leads to disruption of the RNAP elongation complex and recruitment of UvrA. |
| - | *<scene name='2eyq/Domain1a/2'>D1a</scene>
| + | |
| - | *<scene name='2eyq/Domaind2/2'>D2 (UvrB homology module)</scene>
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| - | *<scene name='2eyq/Domain1b/2'>D1b</scene>
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| - | *<scene name='2eyq/Domaind3/2'>D3</scene>
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| - | *<scene name='2eyq/Domain4/2'>D4 (RNAP Interacting Domain)</scene>
| + | |
| - | *<scene name='2eyq/Translocation_module/3'>Translocation module (D5 and D6)</scene>
| + | |
| - | *<scene name='2eyq/Domain7/2'>D7 (handle)</scene>
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| - | <scene name='2eyq/Domainscoloredlabeled/1'>The domains all shown and colored</scene> similar to [http://www.sciencedirect.com/science?_ob=MiamiCaptionURL&_method=retrieve&_udi=B6WSN-4J79KGF-J&_image=fig1&_ba=1&_user=10&_rdoc=1&_fmt=full&_orig=search&_cdi=7051&view=c&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=a4326e4023d32f5de977da04b9a9fdf6 Figure 1B] and [http://www.sciencedirect.com/science?_ob=MiamiCaptionURL&_method=retrieve&_udi=B6WSN-4J79KGF-J&_image=fig2&_ba=2&_user=10&_rdoc=1&_fmt=full&_orig=search&_cdi=7051&view=c&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=b56879d585430e1112f6c5c081a1d158 Figure 2] of [http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6WSN-4J79KGF-J&_user=10&_rdoc=1&_fmt=&_orig=search&_sort=d&_docanchor=&view=c&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=cd0b2f15037bec97859866e118038962 the article describing] the structure.
| + | |
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| - | <scene name='2eyq/Domainscolorsflabe/3'>Domains labeled and colored and displayed similar to</scene> [http://www.sciencedirect.com/science?_ob=MiamiCaptionURL&_method=retrieve&_udi=B6WSN-4J79KGF-J&_image=fig1&_ba=1&_user=10&_rdoc=1&_fmt=full&_orig=search&_cdi=7051&view=c&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=a4326e4023d32f5de977da04b9a9fdf6 Figure 1B] and [http://www.sciencedirect.com/science?_ob=MiamiCaptionURL&_method=retrieve&_udi=B6WSN-4J79KGF-J&_image=fig2&_ba=2&_user=10&_rdoc=1&_fmt=full&_orig=search&_cdi=7051&view=c&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=b56879d585430e1112f6c5c081a1d158 Figure 2] of [http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6WSN-4J79KGF-J&_user=10&_rdoc=1&_fmt=&_orig=search&_sort=d&_docanchor=&view=c&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=cd0b2f15037bec97859866e118038962 the article describing] the structure. <scene name='2eyq/Domainscolorspacefill/1'>Unlabeled</scene>.<br> | + | <ref>DOI:10.1016/j.cell.2005.11.045</ref>. |
| - | <nowiki>[</nowiki>Note: the following view generates a substantial surface which may take several minutes to calculate. Use the one above as an alternative unless you are willing to spend the time.<nowiki>]</nowiki> <scene name='2eyq/Domainssurflabel/1'>Domains labeled and represented much more similar to</scene> [http://www.sciencedirect.com/science?_ob=MiamiCaptionURL&_method=retrieve&_udi=B6WSN-4J79KGF-J&_image=fig1&_ba=1&_user=10&_rdoc=1&_fmt=full&_orig=search&_cdi=7051&view=c&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=a4326e4023d32f5de977da04b9a9fdf6 Figure 1B]. {{Link Toggle LabelsOff}}
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| - | <scene name='2eyq/Translocationmodcontext/1'>The translocation module shown in the context of the Mfd</scene> displayed as in
| + | == References= == |
| - | [http://www.sciencedirect.com/science?_ob=MiamiCaptionURL&_method=retrieve&_udi=B6WSN-4J79KGF-J&_image=fig3&_ba=3&_user=10&_rdoc=1&_fmt=full&_orig=search&_cdi=7051&view=c&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=580f83ffa975d90b5c9d9dc0eb930d8c Figure 3A] of [http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6WSN-4J79KGF-J&_user=10&_rdoc=1&_fmt=&_orig=search&_sort=d&_docanchor=&view=c&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=cd0b2f15037bec97859866e118038962 the article describing] the structure.
| + | <references/> |
| - | | + | |
| - | <scene name='2eyq/Translocation_modhelciasemotif/1'>The seven ATPase/helicase motifs in the translocation domain</scene> are highlighted in red. See figure 3B.
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| - | | + | |
| - | <scene name='2eyq/Walkera/3'>Walker A motif of Mfd</scene> displayed similar to
| + | |
| - | [http://www.sciencedirect.com/science?_ob=MiamiCaptionURL&_method=retrieve&_udi=B6WSN-4J79KGF-J&_image=fig3&_ba=3&_user=10&_rdoc=1&_fmt=full&_orig=search&_cdi=7051&view=c&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=580f83ffa975d90b5c9d9dc0eb930d8c Figure 3B] of [http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6WSN-4J79KGF-J&_user=10&_rdoc=1&_fmt=&_orig=search&_sort=d&_docanchor=&view=c&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=cd0b2f15037bec97859866e118038962 the article describing] the structure.
| + | |
| - | | + | |
| - | <scene name='2eyq/Figure3c/3'>TRG motif and motif VI</scene> indicated similar to [http://www.sciencedirect.com/science?_ob=MiamiCaptionURL&_method=retrieve&_udi=B6WSN-4J79KGF-J&_image=fig3&_ba=3&_user=10&_rdoc=1&_fmt=full&_orig=search&_cdi=7051&view=c&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=580f83ffa975d90b5c9d9dc0eb930d8c Figure 3C] of [http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6WSN-4J79KGF-J&_user=10&_rdoc=1&_fmt=&_orig=search&_sort=d&_docanchor=&view=c&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=cd0b2f15037bec97859866e118038962 the article describing] the structure.
| + | |
| - | </StructureSection> | + | |
| | | | |
| | == 3D Structures of Transcription-repair coupling factor == | | == 3D Structures of Transcription-repair coupling factor == |
The bacterial transcription-repair coupling factor TRCF, also called Mfd translocase, is a DNA repair protein. It has a role in transcription-coupled repair, a subpathway of nucleotide excision repair (NER). Mfd recognizes stalled RNA polymerase (RNAP) and either restarts transcription or removes the stalled polymerase and recruits the NER proteins UvrA and UvrB.
Mfd has ATP hydrolysis activity, DNA binding sites and a UvrA binding sites. These three functions are inhibited in the isolated enzyme, but are activated when Mfd encounters stalled RNA polymerase (or through various mutations that remove inhibitory domains [1]). Mfd also contains an RNA interaction domain (RID) that binds to the beta subunit of RNAP.
| The initial scene shows the domains of Mfd in the conformation of the apo-enzyme. The UvrA interaction site (on domain 2) is occluded by domain 7. The translocase domains (domain 5 and domain 6), through interactions with domains 1 and 3, are locked in an inactive conformation, preventing the typical hinge motion of translocases when they bind and hydrolyze ATP while moving along DNA.
When , the interaction of the RID (domain 4) with RNA polymerase, combined with interactions of domains 5 and domain 6 with DNA and binding to ATP lead to conformational changes that disrupt inter-domain interactions seen in the apo-structure and activate the translocase activity of Mfd. The structures of several intermediates in this process were determined using cryo-EM, shedding light on the activation mechanism, and how translocation of Mfd on DNA leads to disruption of the RNAP elongation complex and recruitment of UvrA.
[2].
References=
- ↑ Selby CP. Mfd Protein and Transcription-Repair Coupling in Escherichia coli. Photochem Photobiol. 2017 Jan;93(1):280-295. doi: 10.1111/php.12675. Epub 2017, Jan 18. PMID:27864884 doi:http://dx.doi.org/10.1111/php.12675
- ↑ Deaconescu AM, Chambers AL, Smith AJ, Nickels BE, Hochschild A, Savery NJ, Darst SA. Structural basis for bacterial transcription-coupled DNA repair. Cell. 2006 Feb 10;124(3):507-20. PMID:16469698 doi:10.1016/j.cell.2005.11.045
3D Structures of Transcription-repair coupling factor
Updated on 15-August-2021
2eyq – EcTRCF – Escherichia coli
6X2N, 6X2F, 6X26, 6X50, 6X43, 6X4W, 6X4Y - EcTRCF, RNA polymerase, RNA, DNA (Cryo EM) />
3hjh – EcTRCF residues 1-470
2b2n - EcTRCF residues 1-333
6yhz - EcTRCF residues 472-547 – NMR
4dfc – EcTRCF D2 domain 127-213 + UvrABC system protein A
6xeo – EcTRCF + DNA – Cryo EM
3mlq – TtTRCF RNA polymerase interacting domain + DNA-directed RNA polymerase subunit β - Thermus thermophilus
6m6a – TtTRCF + RNA polymerase – Cryo EM
6m6b – TtTRCF + RNA polymerase + ATP-γ-S – Cryo EM
2qsr – TRCF C terminal – Streptococcus pneumoni
6ac6, 6aca, 6ac8 – MsTRCF – Mycobacterium smegmatis
6acx – MsTRCF + ADP
Reference
- Deaconescu AM, Chambers AL, Smith AJ, Nickels BE, Hochschild A, Savery NJ, Darst SA. Structural basis for bacterial transcription-coupled DNA repair. Cell. 2006 Feb 10;124(3):507-20. PMID:16469698 doi:10.1016/j.cell.2005.11.045
Created with the participation of Wayne Decatur
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