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Katsuhiko S. MurakamiAssociate Professor of Biochemistry & Molecular Biology 006 Althouse Laboratory, University Park, PA 16802 B.S. in Chemistry, Yamaguchi
University, Japan Murakami Lab Web Site |
Structural Biology of RNA Polymerases
Gene expression is fundamental to all organisms and studying how the genetic code is expressed in molecular terms is critical to cell development and understanding diseases. Our major contribution to biomedical research involves applying structural biology (X-ray crystallography) towards understanding how the genetic information is transcribed into RNA. As an independent investigator at Penn State University since 2003, my laboratory has been studying transcription in bacteriophage N4 and Archaea as model systems for single-subunit and cellular RNAPs, respectively. Major achievements from my laboratory over the past five years, and our ongoing and planned research program are described below.
Models of bacterial RNA polymerase closed (RPc) and open (RPo) complexes with promoter DNA (Science, 2002, 296, 1285-1290).
I. Crystallographic study of the bacteriophage N4 virion encapsulated RNA polymerase
My laboratory has been investigating the transcription mechanism of the bacteriophage N4 virion encapsulated RNA polymerase (vRNAP) by X-ray crystallography. vRNAP is a 3,500 amino acid length uncleaved polyprotein. Its central domain of ~1,100 aa (mini-vRNAP), which possesses the same transcriptional specificity and properties as full-length vRNAP, belongs to the bactriophage T7-like family and also to the A-family of DNA polymerase. The biochemical properties of mini-vRNAP are well characterized by my collaborator, Prof. Rothman-Denes at University of Chicago, and importantly, the enzyme is amenable to crystallization. Therefore, N4 mini-vRNAP is a useful model system for understanding the transcription mechanism of single-subunit RNA polymerases as well as the phosphoryl transfer reaction in both RNA and DNA polymerases. We have made excellent progress on the crystallographic project including: a) the apo-form (Murakami et al., PNAS, 2008); b) the promoter complex (Gleghorn et al., Molecular Cell, 2008); and c) transcription initiation complex structures. This work is supported by NIH/NIGMS.
a) The apo-form mini-vRNAP structure has revealed that it resembles a “fisted right hand” with Fingers, Palm and Thumb sub-domains in the polymerase domain connected to an N-terminal domain. A comparison with the structure of T7 RNA polymerase found that the pathway of the single-stranded template DNA to the active site is blocked by unique structural motifs – plug and motif B loop – in N4 vRNAP indicating that vRNAP must undergo a large-scale conformational change upon promoter DNA binding and explaining the highly restricted promoter specificity of vRNAP.
X-ray crystal structure of N4 mini-vRNAP. Overall views of N4 mini-vRNAP. Domain and subdomains are represented in their characteristic colors. The plug and motif B loop are represented as molecular surfaces with partial transparency. Part of the N-terminal domain (residues 120–145, dark blue) has been identified as interacting with the -11 base of the promoter hairpin triloop; this region is a structural counterpart of the AT-rich recognition loop of the T7 RNAP. (Right) Another N4 mini-vRNAP view, derived from (Left) as indicated by the arrows.
b) Hairpin-stem DNA promoter recognition. The vRNAP recognizes a unique hairpin-form DNA containing 5 bases stem and 3 bases loop as a promoter. Crystal structures of the mini-vRNAP bound to three different promoters provided insights into: 1) how N4 vRNAP structurally accommodates three different types of hairpin-stem DNA promoters; 2) how subtle differences of promoter sequence determine the affinity and salt-stability of the binary complex; and 3) how the N4 polymerase changes its conformation from the transcription inactive apo-form to a transcription-competent binary complex. Despite their low sequence similarity, three out of four promoter recognition motifs are shared with T7 RNA polymerase. Remarkably a totally different mode of promoter generations, hairpin-stem DNA in N4 RNA polymerase and double-stranded DNA in T7 RNA polymerase, are achieved by a quite limited number of specific adaptations in these enzymes.
(Left) Overall structure of the N4 mini-vRNAP bound to the P2 promoter. The promoter DNA hairpin is depicted by a pink ribbon. Domains, sub-domains and structural motifs are labeled. (Right) Positioning of the transcription start site at the vRNAP active site. Residue R318 in the N-terminal domain has a cation-p interaction with base -2 and salt-bridges with the phosphate backbone (depicted by yellow and green dashed lines, respectively) that induce a DNA kink between bases -2 and -1. The +3 base is rotated by ~90º presenting only DNA bases from -1 to +2 to the active site. Amino acid residues essential for activity at the active site are shown: R424 (T/DxxGR motif) for substrate binding; D559 (motif A) and D951 (motif C) for chelating the catalytically essential Mg2+ ions; R666, K670 and Y678 (motif B) for substrate binding. The boxed area is magnified.
II. Crystallographic studies of the archaeal RNA polymerase and transcription
For the past decade, crystal structures of bacterial and eukaryotic RNAPs have revealed new insights into the mechanism of transcription. Structural studies of the archaeal transcription apparatus, however, are just beginning to emerge. Interestingly, archaeal transcription appears to involve a mix of a eukaryotic-like transcription apparatus together with bacteria-like regulatory mechanisms. Our current goal is to determine X-ray crystal structures of archaeal RNAP (~380 kDa, 11 subunits) and its complexes with auxiliary protein factors and nucleic acid. This research is being supported by a grant from the Pew Scholars Program in the Biomedical Sciences since 2005. This year, we reported the first crystal structure of an archaeal RNAP (Hirata et al., Nature, 2008). It revealed striking structural conservation with eukaryotic RNAP II, and highlights the fact that the archaeal transcription system will be an excellent model system for dissecting the molecular basis of eukaryotic transcription. Moreover, our structural and biochemical studies provided the first experimental evidence of any RNAP possessing an iron-sulfur cluster, which raises intriguing questions about the role of iron-sulfur clusters in RNAP structure, function, and regulation.
Currently, we are crystallizing and determining the structures of the archaeal RNAP in a complex with general transcription factors TFB (TFIIB ortholog) and TFE (TFIIEa ortholog), as well as the entire transcription pre-initiation complex including RNAP with TBP-TFB-TFE-promoter DNA. The archaeal pre-initiation complex structure will provide a foundation for studying the eukaryotic Pol II pre-initiation complex (~2.5 MDa complex comprising ~50 polypeptides), which is probably not feasible to determine X-ray crystal structure. In addition, we are crystallizing the backtracked elongation complex, which will represent the first proofreading elongation complex structure determined for any cellular RNAP. This research is being supported by a grant from the Pew Scholars Program in Biomedical Sciences.
Cellular RNAP structures from three domains of life. Surface representation of multi-subunit cellular RNAP structures from Bacteria (left, Thermus aquaticus core enzyme), Archaea (center, S. solfataricus) and Eukarya (right, Saccharomyces cerevisiae Pol II). Each subunit is denoted by a unique color and labeled. Orthologous subunits are depicted by the same color.
III. Genetic studies of the archaeal RNA polymerase subunit and general transcription factors TBP and TFB
To complement the X-ray crystallographic studies, my laboratory is investigating archaeal transcription and regulation using genetic and biochemical approaches. For example, by using gene replacement, we have isolated an archaeal mutant of Thermococcus kodakarensis with the RNAP subunit F-encoding gene (eukaryotic RNAP II subunit Rpb4 ortholog) deleted. This study found that subunit F plays an important role in cell growth at higher temperatures and also is involved in the interaction with TFE, which provides an interesting functional link between archaeal and eukaryotic RNAPs (Hirata et al., Molecular Microbiology, 2008).
The specificity of archaeal transcription is established by two general transcription factors TBP and TFB, and some archaea have multiple genes encoding TBP and/or TFB. However, the biological significance of having multiple general transcription factors in Archaea remains unclear. We have targeted the genes of TBPs in Methanosarcina acetivorans (collaborative project with Prof. Greg Ferry at Penn State University), and TFBs in T. kodakaraensis (collaborative project with Prof. Tadayuki Imanaka at Kyoto University, Japan) in order to understand how multiple combinations of general transcription factors orchestrate their gene expression.
Representative Publications:
- Gleghorn, M.L., Davydova, E.K., Rothman-Denes, L.B. and Murakami, K.S. (2008). Structural basis for DNA-hairpin promoter recognition by the bacteriophage N4 virion RNA polymerase. Mol. Cell. in press.
- Hirata, A., Kanai, T., Santangelo, T.J., Tajiri, M., Manabe, K., Reeve, J.N., Imanaka, T. and Murakami, K.S. (2008). Archaeal RNA polymerase subunits E and F are not required for transcription in vitro, but a Thermococcus kodakarensis mutant lacking subunit F is temperature-sensitive. Mol. Microbiol. 70, 623-633.
- Suharti, K.S. Murakami, S. deVries, and J.G. Ferry (2008). Structural and biochemical characterization of flavoredoxin from the archaeon Methanosarcina acetivorans. Biochemistry. 47, 11528-11535.
- Murakami, K.S., Davydova, E.K. and Rothman-Denes. L.B. (2008). X-ray crystal structure of the polymerase domain of the bacteriophage N4 virion RNA polymerase. Proc Natl Acad Sci U S A, 15, 5046-5051.
- Hirata, A., Klein, B.J. and Murakami, K.S. (2008). The X-Ray Crystal Structure of RNA Polymerase from Archaea. Nature, 451, 851-854.
- Murakami, K. S. and Darst S. A. (2003). Bacterial RNA polymerases: the wholo story. Curr. Opin. Struct. Biol. 13, 31-39.
- Murakami, K. S., Masuda, S. and Darst S. A. (2002). Structural basis of transcription initiation: RNA polymerase holoenzyme at 4 Å resolution. Science 296, 1280–1284.
- Murakami, K. S., Masuda, S., Campbell, E. A., Muzzin, O. and Darst S. A. (2002). Structural Basis of Transcription Initiation: An RNA Polymerase Holoenzyme-DNA Complex. Science 296, 1285-1290.
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