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Crystal Structure of a Bacterial RNA Polymerase Holoenzyme at 2.6 A˚ Resolution

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Crystal Structure of a Bacterial RNA Polymerase Holoenzyme at 2.6 A˚ Resolution articles Crystal structure of a bacterial RNA polymerase holoenzyme at 2.6 A˚ resolution Dmitry G. Vassylyev*†, Shun-ichi Sekine*†, Oleg Laptenko‡, Jookyung Lee‡, Marina N. Vassylyeva†§, Sergei Borukhov‡ & Shigeyuki Yokoyama*†§k * Cellular Signaling Laboratory, and † Structurome Research Group, RIKEN Harima Institute at Spring-8, 1-1-1 Kouto, Mikazuki-cho, Sayo, Hyogo 679-5148, Japan ‡ Department of Microbiology, SUNY Health Science Center, 450 Clarkson Avenue, Brooklyn, New York 11203, USA § RIKEN Genomic Sciences Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan k Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan ........................................................................................................................................................................................................................... In bacteria, the binding of a single protein, the initiation factor j, to a multi-subunit RNA polymerase core enzyme results in the formation of a holoenzyme, the active form of RNA polymerase essential for transcription initiation. Here we report the crystal structure of a bacterial RNA polymerase holoenzyme from Thermus thermophilus at 2.6 A˚ resolution. In the structure, two amino- terminal domains of the j subunit form a V-shaped structure near the opening of the upstream DNA-binding channel of the active site cleft. The carboxy-terminal domain of j is near the outlet of the RNA-exit channel, about 57 A˚ from the N-terminal domains. The extended linker domain forms a hairpin protruding into the active site cleft, then stretching through the RNA-exit channel to connect the N- and C-terminal domains. The holoenzyme structure provides insight into the structural organization of transcription intermediate complexes and into the mechanism of transcription initiation. The DNA-dependent RNA polymerase (RNAP) is the principal exponential growth. The family of related j70 proteins shares four enzyme of the transcription process, and is a final target in many regions of sequence homology, designated 1–4, which are further regulatory pathways that control gene expression in all living divided into several subregions12. The regions 4.2, 2.3–2.4 and 2.5 organisms. In bacteria, RNAP is responsible for the synthesis of were shown to recognize the 235, 210, and the so-called ‘extended all RNAs in the cell (messenger RNA, ribosomal RNA, transfer RNA, 210’ (around nt 217 to 213) elements of the promoter, respect- and so on). The bacterial RNAP exists in two forms: core and ively13–15. In addition, regions 2.3–2.4 are known to interact specifi- holoenzyme. The core enzyme has a relative molecular mass of cally with the 210 region of the nontemplate strand in the open 0 16,17 about 400,000, and consists of five subunits: a-dimer (a2), b, b and promoter complex . Finally, genetic and biochemical data have q. They are evolutionarily conserved in sequence, structure and shown that the portions of j that contact the core include most of function from bacteria to humans1,2. these conserved regions18–20. The transcription cycle in bacterial cells can be divided into three The crystal structures of the Thermus aquaticus RNAP core21, the main stages: initiation, elongation and termination. Although it is yeast Saccharomyces cerevisiae RNAP (polymerase II, Pol II)22, and catalytically active, the core enzyme is incapable of initiating the Pol II ternary elongation complex with a 9-base pair (bp) DNA– transcription efficiently and with specificity. For this, it must bind RNA hybrid23 revealed the high degree of structural similarity an initiation factor, j, to form a holoenzyme that can recognize between the prokaryotic and eukaryotic RNAPs. Together with specific DNA sequences (promoters)3,4. During initiation, the information gained from a wide range of biochemical, biophysical holoenzyme specifically binds to two conserved hexamers in the and genetic studies, these structures allow a plausible modelling of promoter at nucleotide (nt) positions 235 and 210 relative to the bacterial elongation and open promoter transcription com- transcription start site (þ1), to form a closed promoter complex. plexes24,25. Then, it unwinds the double-stranded DNA (dsDNA) around the However, many aspects of the j-dependent transcription 210 region (between nt 212 and þ2), resulting in the open initiation still remain obscure. These include: the promoter recog- promoter complex, and initiates transcription in the presence of nition by the holoenzyme; the formation of a closed promoter nucleoside triphosphate substrates5. After the synthesis of an RNA complex and its transition to an open complex; and the promoter 9–12 nt long, of which 8 or 9 are base-paired with the DNA template escape and dissociation of j factor from RNAP. The high-resolution strand (DNA–RNA hybrid), the transcription complex passes from structure of the T. thermophilus RNAP holoenzyme reported here the initiation to elongation stage6. This transition is characterized by provides information crucial to addressing these questions. It allows the escape of RNAP from the promoter, the dissociation of j from us to identify the structural changes in the core induced by j the core, and the formation of a highly processive ternary elongation binding, and to correlate them with the known functional, biologi- complex. An alternative mechanism has been proposed, in which j cally relevant properties of the holoenzyme. Finally, the holoenzyme factor remains bound to the core in the ternary complex during the structure substantially improves the atomic model of the RNAP elongation stage7,8. During the transition from initiation to core, which is currently available at a modest resolution. elongation, RNAP undergoes a significant conformational change5,9. The j factor has a central role in initiation, being directly Structure determination and overall structure involved in promoter recognition, DNA melting and promoter The holoenzyme crystals belong to the P32 space group with escape and clearance4,10. unit cell dimensions a ¼ b ¼ 236.35, c ¼ 249.04 A˚ . The structure Among several distinct j factors4,11, j70 is primarily responsible was determined by molecular replacement and refined against data for the transcription of most ‘housekeeping’ genes in the cell during to 2.6 A˚ resolution (Fig. 1a, b; see also Supplementary Information). 712 © 2002 Nature Publishing Group NATURE | VOL 417 | 13 JUNE 2002 | www.nature.com/nature articles The holoenzyme maintains the overall crab-claw-like shape N-terminal domain of j, which bridges the b and b 0 pincers of (Fig. 1c) that was observed in the structures of the T. aquaticus the claw on the upstream DNA side of RNAP, creating a wall that core and S. cerevisiae Pol II21,22. However, the presence of the j70 blocks the opening of the DNA-binding channel; (2) a hairpin-like subunit and the large portion of the nonconserved N-terminal motif of j, which protrudes into the cleft of the active site, towards domain of the b 0 subunit in the model greatly extends the b 0 pincer, the possible location of the RNA–DNA hybrid; and (3) an extended thus making the analogy to a crab claw even more fitting. The j70 fragment following the hairpin-like motif, which occupies the subunit is located almost entirely on the core surface, except for RNA-exit channel. ashortsegment(j313–342), which is buried within the core The core-to-holoenzyme conversion is accompanied by changes molecule. The binding of j to the RNAP core does not seem to in all structural domains of the core enzyme. The most noticeable result in the widening of the DNA-binding channel, as was pre- include the bG flap domain (b702–828), the aI subunit, the large viously thought. On the contrary, certain structural elements of j N-terminal portion of b (b20–395), and the b 0 coiled-coil domain reduce the available space of functionally important cavities in (b 0 540–585) (Fig. 2). The RNA-exit channel is most significantly RNAP that are presumed to accommodate the transcription bubble affected by j70 binding. The channel is constricted by the b flap and the RNA product. These elements include: (1) the second domain, which is shifted by about 5–6 A˚ towards the j C-terminal domain compared with the T. aquaticus core21. The change in the orientation of the ‘flap tip’ helix, which is now squeezed between three a-helices of j (Fig. 2), is even more dramatic (,11 A˚ shift). The change in the orientation of individual domains of the RNAP subunits (aI and b20–395) that are adjacent to the b flap domain seems to mirror the flap domain rotation. In the crystal structure, we observed many Mg2þ ions bound to holoenzyme, most of which formed a coat on the protein surface. These metal ions could be important in the binding and bending of the DNA molecule, which is proposed to be wrapped around RNAP during transcription26. Structure of j70 The atomic model of the j70 subunit lacks the disordered N- terminal domain (j1–73), which includes the conserved j region 1.1. This is a self-inhibitory domain, which is known to mask the DNA-binding regions of j before it binds the core27. Its precise role in transcription initiation is not understood. The modelled structure of j70 consists entirely of a-helices connected by either turns or loops (Fig. 3). It can be divided into four structural domains: N-terminal domain 1 (ND1), N-terminal domain 2 (ND2), ‘linker’ domain (LD) and C-terminal domain (CD). ND1 consists of eight a-helices (j74–254) comprising four helix– turn–helix (HtH) motifs: HtH1 (j75–121), HtH2 (j94–137), HtH3 (j123–147) and HtH4 (j152–203) (Figs 3 and 4a). This domain encompasses region 1.2 up to the N-terminal half of region 2.4, including the nonconserved segment between regions 1 and 2. ND1 has a U-shaped structure, and is connected to ND2 (j261–312) by a short linker loop (j255–260), which lies at the C terminus of region 2.4.
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