RNA Polymerase II with Transcription Factor IIB

Jason Cinti ('16) and Issam Hamdallah ('16)


I. Introduction

     In order for a gene to be expressed, it must first be transcribed into mRNA, the precursor of all proteins. The enzyme most responsible for the synthesis of mRNA is RNA polymerase II (Pol II). By itself, Pol II is incapable of binding upstream promoters and beginning the synthesis of mRNA. Instead, a multitude of transcription factors (TFIIB, IID, IIE, and IIH) assemble at the promoter DNA to recruit and properly place Pol II. Together, this large complex of proteins, called the pre-initiation complex (PIC), ensure accurate initiation because each contributing element must bind in a specific order and in a precise arrangement before transcription can occur. 
    Of the various components of the PIC, the TATA-box binding protein (TBP) and transcription factor IIB (B) are responsible for the successful delivery of Pol II to the promoter DNA as well as promoter clearance. Specifically, B stabilizes TBP to the TATA-element DNA to form the ternary complex, which in turn allows RNA polymerase II to bind. Once RNA polymerase II binds to the ternary complex, recruitment of the additional transcription factors to form the complete PIC is made possible. Here we examine the structure of B and how B interacts with various components of Pol II.


II. General Structure of B:

          The polypeptide chain is composed of four key domains . At the N-terminus resides the B-ribbon. The B-ribbon binds to the dock domain of Pol II and is composed of two antiparallel beta sheets and a pocket for a zinc ion . A pair of cysteine residues (24 and 27, and 45 and 47) share a bond with the zinc ion at an average distance of 0.243 nm. The next domain along the protein chain towards the C-terminus is the B-reader , aptly named for its role in reading the DNA sequence during the selection of a transcription start site (TSS). The B-reader contains a single alpha helix that spans from residues 57-69 and loops to the B-linker domain . The B-linker can be found in the rudder and clamp coiled-coil domain of Pol II and is composed of one beta strand (residues Asn 71 - Gly 86) and an alpha helix (residues Thr 87 - Ser 110). Following the B-linker is the B-core , which plays a key role in anchoring the TBP to the TATA-element DNA and thus stabilizes the ternary complex. The B-core is composed of an N-terminues cyclin region and a C-terminus cyclin region connected by a small linker region . Each cyclin fold is composed of five similar alpha helices, yet the two folds differ because of the presence of a short alpha helix on the most outward region of the C-terminus fold . The B-core is able to anchor the TBP to the TATA-box by interacting with both the DNA backbone and the TBP. B-core binding to the DNA backbone occurs non-specifically and is mediated by positively charged residues that can interact with the negatively charged DNA backbone both upstream and downstream of the TATA-box . The Coulombic attraction between the B-core and the DNA is crucial to the stability of the TBP, TATA-box and B ternary complex. Further stabilization of this complex can be attributed to the interaction between the B-core and TBP . It is a single loop in the C-terminus of TBP that interacts with the positively charged residues of the B-core . Many of the residues form hydrogen bonds and Van der Waal interactions with the B-core to stabilize the complex, but the sulfide bond between residues Lys-188 and Glu-146 particularly dominates the interaction .


III. Interactions with Pol II domains:

          As soon as B secures TBP to the TATA-box on DNA, RNA polymerase II can recognize the complex and bind. The zinc ion in the B N-terminus creates a ribbon like fold pocket - a structure very conducive to forming strong interactions with other proteins. This pocket docks the B polypeptide chain at a single beta strand in Pol II located in a small domain called the dock domain . Disruptions in the zinc ribbon fold caused by mutations often result in decreased levels of transcription, suggesting that the specific folds created by the zinc ion facilitates easier recruitment of Pol II. The B-ribbon residues Glu-40, Leu-47, and Glu-26 are a few of many that help stabilize the initial recruitment of Pol II by interacting with dock domain residues Asp-414, Ala-412, and Asp-411, respectively . B then threads through a region of Pol II called the RNA exit tunnel and the B-reader makes contacts with the active center . The active center contains a magnesium ion and is the primary location of messenger RNA catalysis. Proximate to the active center, the B-linker domain is found at the the rudder and clamp coiled coil domain , which is responsible for positioning the DNA during initiation and maintaining the proper transcription bubble conformation. Finally, the B-core pushes up against the hybrid binding / wall domain , which plays a key role in the elongation process by binding nascent mRNA as it is generated. With Pol II secured to B , both of which are securlely anchored to TATA-element DNA, the additional transcription factors can bind to form the PIC and transcription can proceed. 


V. References

(1) Bushnell D.A., Westover K. D., Davis R.E., Kornberg R.D. 2003. Structural Basis of Transcription: An RNA Polymerase II?TFIIB Cocrystal at 4.5 Angstroms. Science 303: 983-988

(2) Liu X., Bushnell D.A., Wang D., Guillermo C., Kornberg R.D. 2010. Structure of an RNA Polymerase II-TFIIB Complex and Transcription Initation Mechanism. Science 327: 206-209

(3) Hahn S., Roberts S. 2000. The zinc ribbon domains of the general transcription factors TFIIB and Brf: conserved functional surfaces but different roles in transcription initiation. Genes and Development 14: 719-730

(4) Kostrewa D., Zeller M. E., Armache K. J., Seizl M., Leike K., Thomm M., Cramer P. 2009. RNA polymerase II?TFIIB structure and mechanism of transcription initiation. Nature 462: 323-330

(5) Nikolov, D.B., Chen, H., Halay, E.D., Usheva, A.A., Hisatke, K., Kun Lee, D., Roeder, R.G., Burley., S.K., Crystal structure of a TFIIB-TBP-TATA-element ternary complex. Nature 377, 1190128

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