Katsuhiko S. Murakami Assistant Professor of Biochemistry & Molecular Biology 006 Althouse Laboratory, University Park, PA 16802 Phone: (814) 865-2758 Fax: (814) 865-2759 Email: kum14@psu.edu B.S. in Chemistry, Yamaguchi University, Japan Ph.D. in Genetics, National Institute of Genetics, Japan Post-doctoral work in Structural Biology, The Rockefeller University Murakami Lab Web Site

Structural Biology of RNA Polymerases

Transcription is the major control point of gene regulation and RNA polymerase is the central enzyme of transcription. The long-term goal of this laboratory is to understand the mechanism of transcription and its regulation. Determining the three- dimensional structures of RNA polymerase and transcription complexes is essential. We are particularly interested in determining X-ray crystallographic structures of RNA polymerases from different kind of species: bacteriophage (single-unit RNA polymerase), bacteria and archaea (multi-subunit cellular RNA polymerase), and influenza virus (RNA-dependent RNA polymerase). In addition, we try to determine high resolution structures of RNA polymerase complex with DNA, RNA and regulatory factors. Based on the structures, we study the detailed functions of RNA polymerase using biochemical and biophysical methods. The ultimate goal in the structural effort is to solve all structures at different steps of transcription process (promoter recognition, transcription initiation, elongation and termination).

Models of bacterial RNA polymerase closed (RPc) and open (RPo) complexes with promoter DNA (Science, 2002, 296, 1285-1290).

X-ray crystallographic studies of Bacteriophage N4 RNA polymerase

Coliphage N4 virion RNA polymerase (vRNAP), which is injected into the host upon infection, transcribes the phage early genes from promoters that have a five base pair stem-three nucleotide loop hairpin structure. We determined the 2.0 Å resolution X-ray crystal structure of N4 mini-vRNAP, a member of the T7-like, single-unit RNAP family and the minimal component having all RNAP functions of the full-length vRNAP. The structure resembles a “fisted right hand” with Fingers, Palm and Thumb sub-domains connected to an N-terminal domain. We established that the specificity loop extending from the Fingers along with Trp129 of the N-terminal domain play critical roles in hairpin-promoter recognition. A comparison with the structure of the T7 RNAP initiation complex reveals that the pathway of the DNA to the active site is blocked in the apo-form vRNAP, indicating that vRNAP must undergo a large-scale conformational change upon promoter DNA binding and explaining the highly restricted promoter specificity of vRNAP that is essential for phage early transcription. This work is supported by NIH/NIGMS.

  X-ray crystal structure of N4 mini-vRNAP

The X-Ray Crystal Structure of RNA Polymerase from Archaea

The transcription apparatus in Archaea can be described as a simplified version of its eukaryotic RNA polymerase (Pol II) counterpart, comprising a Pol II-like enzyme as well as two general transcription factors, the TATA-binding protein (TBP) and the eukaryotic TFIIB ortholog TFB. It has been widely understood that precise comparisons among cellular RNA polymerase crystal structures could reveal structural elements common to all enzymes and that these insights would be useful to analyze components of each enzyme that enable it to perform domain-specific gene expression. However, the structure of archaeal RNA polymerase has been limited to individual subunits. We determined the first crystal structure of the archaeal RNA polymerase from Sulfolobus solfataricus at 3.4 Å resolution, completing the suite of multi-subunit RNA polymerase structures from all three domains of life. We also report the high resolution (at 1.76 Å) crystal structure of the D/L subcomplex of archaeal RNA polymerase and provide the first experimental evidence of any RNA polymerase possessing an iron-sulfur (Fe-S) cluster, which may play a structural role in a key subunit of RNA polymerase assembly. The striking structural similarity between archaeal RNA polymerase and eukaryotic Pol II highlights the simpler archaeal RNA polymerase as an ideal model system for dissecting the molecular basis of eukaryotic transcription. This work is supported by the Pew Scholars Programs 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.

Representative Publications:

• 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. submitted. .
• Hirata, A., Klein, B.J. and Murakami, K.S. (2008). The X-Ray Crystal Structure of RNA Polymerase from Archaea. Nature, in press.

• Murakami, K. S, Masuda. S and Darst, S. A. (2003). Crystallographic analysis of Thermus aquaticus RNA polymerase holoenzyme and a holoenzyme/promoter DNA complex. Methods Enzymol. 370, 42-53.
• 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.

Search the MEDLINE database at PubMed for articles by K Murakami