| dc.description.abstracteng | Chapter 1
RNA polymerase II (Pol II) is the central enzyme that transcribes eukaryotic protein-coding genes to produce mRNA. The mushroom toxin α-amanitin binds Pol II and inhibits transcription at the step of RNA chain elongation. Pol II from yeast binds α-amanitin with micromolar affinity, whereas metazoan Pol II enzymes exhibit nanomolar affinities. Here, I present the high resolution cryo-EM structure of α-amanitin bound to and inhibited by its natural target, the mammalian Pol II elongation complex. The structure revealed that the toxin is located in a pocket previously identified in yeast Pol II but forms additional contacts with metazoan-specific residues, which explains why its affinity to mammalian Pol II is ∼3000 times higher than for yeast Pol II. The work provides the structural basis for the inhibition of mammalian Pol II by the natural toxin α-amanitin and highlights that cryo-EM is well suited to studying interactions of a small molecule with its macromolecular target.
Chapter 2
Transcription termination is coupled to pre-mRNA 3’ processing. In mammals, more than twenty protein factors are involved in these processes. The definition of the cleavage site needs not only protein factors but also specific cis sequence elements on pre-mRNA. The best known cis elements include the polyadenylation signal (PAS, featuring the base sequence AAUAAA), the upstream elements (USE, featuring the base sequence UGUA) and downstream elements (DSE, characteristically GU/U rich), which are bound by the cleavage and polyadenylation (CPSF) complex, the cleavage factor I (CFI) complex and the cleavage stimulation factor (CstF) complex respectively. Other termination/3’ processing factors include cleavage factor II (CFII), polyadenylation polymerase (PAP), polyadenylate-binding nuclear protein 1 (PABPN1), Pol II carboxy terminal domain (CTD), symplekin (SYMPK), as well as some kinases and phosphatases and other factors. Based on the functional differences, CPSF complex is divided into two modules: the polymerase module, which is composed of CPSF160, WDR33, CPSF30 and Fip1, and the nuclease module which is composed of CPSF100 and CPSF73. The polymerase module binds specifically to PAS site while the nuclease module is responsible for the cleavage of pre-mRNA. CPSF73 is the endonuclease. After cleavage, a polyadenylic acid tail (poly(A) tail) is added to the 3’ end of RNA by PAP. In this work, I managed the expression and purification of the sub-complexes CFI, CFII, CstF, CPSF polymerase module and CPSF nuclease module plus symplekin. The CstF complex can be crystallized but the diffraction of the crystal was not good enough to solve the structure yet. The CPSF polymerase module and nuclease module plus symplekin can form a stable complex which is suitable for cryo-EM structure analysis. From the initial data processing, the extra density in addition to the polymerase module can be seen. However, the resolution of the density map needs to be improved by further processing. | de |