Structural Molecular Biology-A Personal Reflection on The

Commentary

Structural Molecular Biology—A Personal Reflection on the Occasion of John Kendrew's 100th Birthday

Patrick Cramer

Max Planck Institute for Biophysical Chemistry, Department of Molecular Biology, Am Fassberg 11, 37077 Göttingen, Germany http://dx.doi.org/10.1016/j.jmb.2017.05.007 Edited by P Wright

Abstract

Here, I discuss the development and future of structural molecular biology, concentrating on the eukaryotic transcription machinery and reflecting on John Kendrew's legacy from a personal perspective. © 2017 Elsevier Ltd. All rights reserved.

Every structural biologist knows how special it Kendrew did much to establish EMBL and served as feels to obtain the three-dimensional structure of a its first Director-General. At EMBL, I started to work protein that one so desperately wishes to under- on gene transcription [4,5] and continued at Stanford stand. How then must have John Kendrew felt when University as a postdoctoral fellow with Roger he solved the first of all protein structures? In 1957, Kornberg, who was trained at the Laboratory of his team determined the structure of myoglobin, the Molecular Biology, like many others. We obtained protein that stores oxygen in muscle [1]. Kendrew major insights into the transcription process, which is visualized the electron density as a set of contour central to the regulation of genes during cellular maps, drawn on stacked plastic sheets, and arrived responses and organism development. at the first protein model. When I teach, I often show the beautiful black-and-white photograph of RNA polymerase structure Kendrew standing beside the large myoglobin model. The model contained cylindrical portions Around the millennium, we were able to solve the that provided evidence for the existence of α-helices structure of the central enzyme of transcription in predicted by Linus Pauling et al. [2]. Kendrew was eukaryotic cells, RNA polymerase II (Pol II) [6,7].I lucky that myoglobin consisted of helices because obtained advice and help from colleagues in the these can be resolved at low resolution. Although the Kornberg lab, in particular David Bushnell, Avi Gnatt, existence of helices was expected, it came as a and Jianhua Fu [8]. Structure determination relied on surprise that a protein could have such a unique and phasing by multiple isomorphous replacement [6],a defined structure. This suggested fundamental method that Perutz and colleagues had pioneered. I biological concepts for macromolecular recognition remember that the final phasing run took several and enzymatic catalysis. At that time, it was certainly weeks on a high-end computer workstation. Already impossible to imagine the splendid variety of protein in the 1950s, Kendrew carried out Fourier transforms structures to be unveiled. on a very early computer, EDSAC-2. He envisioned Although I never met John Kendrew, it feels as if in his Nobel lecture that much faster computers we crossed paths. When I conducted my first crystal would be required. Indeed, we can now calculate structure analysis at Cambridge in 1994 [3], I got to electron densities on laptop computers within know many of those who had worked with Kendrew minutes. when he headed the Structural Biology Division, It was so pleasing to see the intricate structure of including Max Perutz, the first director of the MRC Pol II, in free form [6,7] and with bound DNA and Laboratory of Molecular Biology. A year later, I RNA [9]. The structure revealed 10 protein subunits became a PhD student at the European Molecular and later all 12 subunits [10–12], which formed a Biology Laboratory (EMBL) and learned that John “marvelous machine for making messages”—as

Fig. 1. Current view of transcription of a eukaryotic gene by RNA polymerase II based on results from integrated structural biology. Current structures of the Pol II initiation complex (left), the Pol II elongation complex (middle), and the nucleosome (right) are shown. DNA is in blue and cyan, Pol II is in silver, initiation and elongation factors are in different colors, the coactivator complex Mediator is in light blue, and a histone octamer is in gold. The figure is based on PDB entries 1WCM, 5FZ5, 1AOI, 3PO3, and 2EXU, and EMDB entries 3305, 8131, and 8307.

noted by Aaron Klug [13], another colleague of movie of the nucleotide addition cycle [25]. The movie Kendrew. RNA polymerase structures had also been animates the process DNA template-dependent solved from bacteria by the laboratory of Seth Darst RNA chain elongation by combining static structural [14] and from the bacteriophage T7 by the laboratory pictures and morphing between them. We later of Tom Steitz [15], who was also a postdoctoral fellow extended the movie [26], making use of structural at the Laboratory of Molecular Biology. Comparisons studies that positioned transcription factors on Pol II with the eukaryotic Pol II structure revealed that [27–30]. The extended movie included DNA loading, multisubunit cellular polymerases share a conserved RNA proofreading, and Pol II backtracking. Crystallo- core that holds the active center [16]. The single- graphic studies of Pol II complexes also elucidated subunit T7 polymerase was substantially different, but how the enzyme deals with DNA lesions and DNA, RNA, and substrate nucleoside triphosphates modifications [31–34]. were arranged in a similar way [17].Itfeltlikea The movie lacked information about how RNA privilege to glimpse the RNA polymerase structure, synthesis initiates at the beginning of genes. The one of nature's well-kept secrets. structural basis for transcription initiation was studied for bacterial RNA polymerase, which requires a single initiation factor, the sigma factor, to bind and open From snapshots to movies promoter DNA [35–38]. Pol II initiation, however, of transcription involves the assembly of around 70 polypeptides and was only recently elucidated [39–41].Modelsofthe The polymerase structure was the starting point closed and open promoter complexes could be for a decade-long process of elucidating the intrica- obtained based on crystallographic studies of the cies of gene transcription (Fig. 1). Returning to Europe Pol II complex with the initiation factor TFIIB [28], in 2001, I established a laboratory at the Gene Center which used structural information of how TFIIB binds of the University of Munich. There, we determined DNA [42]. For such crystal structure determinations, a crystal structures of Pol II complexes with nucleic new trick or twist had to be found each time [43].Itthus acids [18–21]. From those efforts and complementary became clear that it would be difficult to solve larger results from colleagues [22–24], we prepared a first and transient Pol II assemblies with the use of Download English Version: https://daneshyari.com/en/article/5533262

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