Transcription-repair coupling factor

From Proteopedia

Revision as of 14:42, 16 August 2021

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.

Function

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.

Relationship to other enzymes

The N-terminal part of Mfd shows sequence similarity to UvrB, including in the domain of UvrB that interacts with UvrA. However, the conserved helicase motifs present in UvrB (responsible for binding and hydrolyzing ATP) are absent in that part of Mfd. Furthermore, the sequence segment known to fold as a beta hairpin in UvrB (involved in clamping down a single strand of DNA) seems absent. The C-terminal part of Mfd shows sequence similarity to SF1/SF2 helicases (UvrB is an example), containing conserved helicase/translocase motifs.

Structure and conformational change

spinqualitylabelsall models Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image The initial scene (switch to ) shows the domains of Mfd in the conformation of the apo-enzyme.[2] 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 Mfd binds to DNA in the presence of ADP AlFx, Mfd undergoes large domain rearrangements, giving a hint how it might bind to stalled RNAP.[3]. A first glimpse of the Mfd RNAP complex was through cryo-EM, but large parts of the Mfd protein were not resolved.[4] In 2021, the structures of several intermediates in the presences of stalled RNAP were determined using cryo-EM, shedding light on the loading and activation mechanism, as well as how translocation of Mfd on DNA leads to disruption of the RNAP elongation complex and recruitment of UvrA.[5] When , the RID (domain 4) binds to the beta subunit of RNA polymerase and domains 5 and domain 6 bind to DNA and ATP. These multiple binding events require a different relative orientation of RID and the translocase domains, and the necessary conformational changes disrupt inter-domain interactions seen in the apo-structure while activating the translocase activity of Mfd. Once the translocase domains are activated, they move closer and closer to the surface of the RNA polymerase in multiple cycles of ATP hydrolysis until . The translocase moving along the DNA together with the RID tethered at a fixed point on the RNAP surface result in substantial reorganization of the domain contacts and relative orientation. Eventually, this leads to the the , thus recruiting the NER machinery to the damaged template strand for subsequent repair. Further translocation (not shown) will lead to distabilization of the RNAP: nucleic acid complex, helping to dislodge the stalled RNAP. The changing domain contacts are schematically summarized below. Distinct domain contacts in various Mfd structures One of the functions of the large conformational change is to create a topological lock around the DNA to support translocation in a highly processive manner. Whereas in other systems, motor protein, clamp and clamp loader are separate proteins, all these functions are housed here in a single polypeptide. Mfd uses a single ATPase activity to create a sliding clamp and load it onto the DNA. The necessary reorganization of domains is visualized in a . [6] In the apo-structure, domains 3 and 4 are far apart from each other (connected by an extended linker), but they make direct contacts when Mfd is loaded on DNA. On the other hand, domains 4 and 5 make direct contacts in the apo-structure, but are far apart from each other (coonected by an extended linker) when Mfd is loaded on DNA. Finally, domain 2 and domain 7 bind to each other in the apo-state whereas domain 2 is available for binding UvrA in the DNA-bound state.

References

1. ↑ 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
2. ↑ 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
3. ↑ Brugger C, Zhang C, Suhanovsky MM, Kim DD, Sinclair AN, Lyumkis D, Deaconescu AM. Molecular determinants for dsDNA translocation by the transcription-repair coupling and evolvability factor Mfd. Nat Commun. 2020 Jul 27;11(1):3740. doi: 10.1038/s41467-020-17457-1. PMID:32719356 doi:http://dx.doi.org/10.1038/s41467-020-17457-1
4. ↑ Shi J, Wen A, Zhao M, Jin S, You L, Shi Y, Dong S, Hua X, Zhang Y, Feng Y. Structural basis of Mfd-dependent transcription termination. Nucleic Acids Res. 2020 Nov 18;48(20):11762-11772. doi: 10.1093/nar/gkaa904. PMID:33068413 doi:http://dx.doi.org/10.1093/nar/gkaa904
5. ↑ Kang JY, Llewellyn E, Chen J, Olinares PDB, Brewer J, Chait BT, Campbell EA, Darst SA. Structural basis for transcription complex disruption by the Mfd translocase. Elife. 2021 Jan 22;10. pii: 62117. doi: 10.7554/eLife.62117. PMID:33480355 doi:http://dx.doi.org/10.7554/eLife.62117
6. ↑ The Storymorph Jmol scripts were used to create the interpolation shown in the morph. Coordinates available on Proteopedia

3D Structures of Transcription-repair coupling factor

Updated on 16-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

Created with the participation of Wayne Decatur