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.
FunctionFunction
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 enzymesRelationship 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 changeStructure and conformational change
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 further 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.  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. 3D Structures of Transcription-repair coupling factorTranscription-repair coupling factor 3D structures References
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Created with the participation of Wayne Decatur