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J. Biol. Chem., Vol. 281, Issue 26, 18216-18226, June 30, 2006

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Structural, Biochemical, and Dynamic Characterizations of the hRPB8 Subunit of Human RNA Polymerases^*

Xue Kang, Yunfei Hu, You Li^¶, Xianrong Guo^¶, Xiaolu Jiang^¶, Luhua Lai^¶, Bin Xia^¶, and Changwen Jin^¶1

From the Beijing Nuclear Magnetic Resonance Center, the ^¶College of Chemistry and Molecular Engineering, and the College of Life Sciences, Peking University, Beijing 100871, China

The RPB8 subunit is present in all three types of eukaryotic RNA polymerases and is highly conserved during evolution. It is an essential subunit required for the transcription of nuclear genes, but the detailed mechanism including its interactions with different subunits and oligonucleotides remains largely unclear. Herein, we report the three-dimensional structure of human RPB8 (hRPB8) at high resolution determined by NMR spectroscopy. The protein fold comprises an eight-stranded -barrel, six short helices, and a large unstructured -loop. The overall structure of hRPB8 is similar to that of yRPB8 from Saccharomyces cerevisiae and belongs to the oligonucleotide/oligosaccharide-binding fold. However, several features of the tertiary structures are notably different between the two proteins. In particular, hRPB8 has a more clustered positively charged binding interface with the largest subunit RPB1 of the RNA polymerases. We employed biochemical methods to detect its interactions with different single-stranded DNA sequences. In addition, single-stranded DNA titration experiments were performed to identify the residues involved in nonspecific binding with different DNA sequences. Furthermore, we characterized the millisecond time scale conformational flexibility of hRPB8 upon its binding to single-stranded DNA. The current results demonstrate that hRPB8 interacts with single-stranded DNA nonspecifically and adopts significant conformational changes, and the hRPB8/single-stranded DNA complex is a fast exchanging system. The solution structure in conjunction with the biochemical and dynamic studies reveal new aspects of this subunit in the molecular assembly and the biological function of the human nuclear RNA polymerases.

Received for publication, December 13, 2005 , and in revised form, April 19, 2006.

The atomic coordinates and structure factors (code 2F3I) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported by National Natural Science Foundation of China Grants 30125009 (to B. X.) and 30325010 (to C. J.) and in part by Grant 2004CB520801 from the National Key Basic Research Program of China. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental figures.

¹ To whom correspondence should be addressed: Beijing Nuclear Magnetic Resonance Center, Peking University, Beijing 100871, China. Tel.: 86-10-6275-6004; Fax: 86-10-6275-3790; E-mail: changwen{at}pku.edu.cn.

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