NAME: Florian Brueckner
AFFILIATION: University of Munich, Germany
CONTACT: brueck@lmb.uni-muenchen.de
TITLE: "Structural and Functional Studies of the RNA Polymerase II Elongation Complex"
ABSTRACT: Florian Brueckner and Patrick Cramer, Gene Center, University of Munich, Feodor-Lynen-Str. 25, D-81377 Muenchen
RNA Polymerase II (Pol II) transcribes all protein-coding genes in eukaryotic cells, synthesising messenger RNA. The transcription cycle consists of three stages: initiation at a promoter, elongation and termination.
Structural studies on Pol II from Saccharomyces cerevisiae were initiated in the Kornberg lab with the core enzyme consisting of 10 different protein subunits with a total molecular weight of 469 kDa. Dehydration of the crystals using a soaking procedure shrank the unit cell and improved diffraction from 6 to below 3 Å resolution. Phase information was obtained from multiple heavy atom derivatives including a six-tantalum-atom cluster [1]. A refined atomic model of the core enzyme was obtained at 2.8 Å [2].
Crystals of the complete 12-subunit Pol II (in total 514 kDa) including the two additional subunits Rpb4 and Rpb7 were then obtained but displayed a high solvent content of 80 % and diffracted only to around 4 Å [3]. To obtain an atomic model of the complete Pol II, atomic models of the core Pol II (2.8 Å) and of the additional subcomplex Rpb4/Rpb7 (2.3 Å) were combined and refined against diffraction data from a complete Pol II crystal at 3.8 Å resolution [4].
Crystals of the complete Pol II elongation complex could be obtained by cocrystallizing complete 12-subunit Pol II with a pre-annealed synthetic nucleic acid scaffold consisting of template DNA, nontemplate DNA and product RNA under crystallization conditions favouring protein-nucleic acid interactions. The structure at 4.0 Å clearly revealed the topology of the nucleic acids inside the enzyme and suggested mechanisms for DNA-DNA and DNA-RNA strand separation. Additionally, the binding of a substrate NTP to the active centre and the influence of the elongation factor TFIIS were investigated [5].
In a follow-up study, elongation complexes with thymine-thymine photodimers at specific locations in the template strand were designed. The recognition of these bulky DNA lesions by Pol II is the first step of transcription coupled DNA repair and is therefore a prerequisite for the efficient removal of these lesions. A set of crystal structures (3.8 - 4.0 Å) and in-vitro transcription assays established the mechanism that underlies lesion recognition. Unexpectedly, Pol II specifically incorporates a uracil opposite the 3’-thymine of the photodimer and the resulting mismatch is crucial for lesion recognition. Furthermore, our structural results suggest non-allosteric recruitment of repair factors [6].
The ability to assemble and study tailor-made elongation complexes allows us to address many more open questions on transcription elongation, concerning e.g. the recognition of other kinds of DNA damages, transcriptional mutagenesis, fidelity, or the mechanism of nucleotide addition and translocation. Optimization of the crystals, the data collection equipment as well as data processing enables extension of the resolution limit towards 3 Å.
1. P. Cramer et al., Science 288, 640-649 (2000)
2. P. Cramer et al., Science 292, 1863-1876 (2001)
3. K.-J. Armache et al., PNAS 100, 6964-6968 (2003)
4. K.-J. Armache et al., J. Biol. Chem. 280, 7131-7134 (2005)
5. H. Kettenberger et al., Mol. Cell 16, 955-965 (2004)
6. F. Brueckner et al., Science 315, 859-862 (2007)