Structure of the human Mediator-bound transcription preinitiation complex.

Area of Science:

• Molecular Biology
• Structural Biology
• Biochemistry

Background:

• Eukaryotic transcription involves RNA polymerase II (Pol II) and general transcription factors forming a preinitiation complex (PIC).
• The Mediator coactivator is essential for PIC assembly and stimulates Pol II C-terminal domain (CTD) phosphorylation by CDK7, a TFIIH subunit.

Purpose of the Study:

• To determine the high-resolution cryo-electron microscopy structure of the human Mediator-bound PIC.
• To elucidate the structural basis of Mediator and TFIIH interactions within the PIC.
• To provide insights into the mechanism of Pol II CTD phosphorylation.

Main Methods:

• Cryo-electron microscopy (cryo-EM) was used to obtain the structure of the human Mediator-bound PIC.
• The structure was resolved to a resolution below 4 angstroms.
• Analysis of protein-protein interactions and binding sites within the complex.

Main Results:

• The cryo-EM structure reveals flexible tethering of transcription factor binding sites to the Mediator tail module.
• CDK7 is stabilized through multiple contacts with Mediator.
• Two distinct binding sites for the Pol II CTD were identified: one within Mediator and another in the CDK7 active site.

Conclusions:

• The structure provides direct evidence for Pol II CTD phosphorylation occurring within the Mediator-bound PIC.
• The findings illuminate the roles of Mediator and CDK7 in regulating transcription initiation.
• This work offers a detailed molecular understanding of a key step in eukaryotic gene expression.