RNA polymerase and transcription elongation factor Spt4/5 complex structure

Edited by* Roger D. Kornberg, Stanford University School of Medicine, Stanford, CA, and approved November 30, 2010 (received for review September 15, 2010)
December 27, 2010
108 (2) 546-550


Spt4/5 in archaea and eukaryote and its bacterial homolog NusG is the only elongation factor conserved in all three domains of life and plays many key roles in cotranscriptional regulation and in recruiting other factors to the elongating RNA polymerase. Here, we present the crystal structure of Spt4/5 as well as the structure of RNA polymerase-Spt4/5 complex using cryoelectron microscopy reconstruction and single particle analysis. The Spt4/5 binds in the middle of RNA polymerase claw and encloses the DNA, reminiscent of the DNA polymerase clamp and ring helicases. The transcription elongation complex model reveals that the Spt4/5 is an upstream DNA holder and contacts the nontemplate DNA in the transcription bubble. These structures reveal that the cellular RNA polymerases also use a strategy of encircling DNA to enhance its processivity as commonly observed for many nucleic acid processing enzymes including DNA polymerases and helicases.
Transcription by RNA polymerase (RNAP) plays a central role in gene expression, and this process is highly regulated at many steps, including promoter recognition, transcription activation, elongation, and termination. During transcription, several transcription elongation factors communicate with RNAP and regulate the velocity of RNA synthesis, transcriptional pausing, and termination (1). Recent genome-wide analyses of transcription elongation revealed the importance of transcription elongation in the regulation of gene expression (24). For example, a large fraction of transcribing eukaryotic RNAP II (Pol II) pauses near promoters to facilitate rapid changes in gene expression during cell development.
Bacterial NusG is perhaps the best characterized transcription elongation factor in vivo and in vitro. Diverse functions of NusG have been reported, e.g., Escherichia coli NusG reduces RNAP pausing and intrinsic termination (5, 6), whereas Bacillus subtilis and Thermus thermophilus NusG enhance pausing (7, 8). NusG consists of the NusG amino-terminal (NGN) domain and Kyprides–Onzonis–Woese (KOW) motif at the C-terminal domain (hence also called KOW domain), and these two domains fold independently and are connected by a flexible linker of 13 amino acids (9, 10) (Fig. 1B and Fig. S1C). The NGN domain has been assigned the function for regulating transcription elongation. The KOW domain, on the other hand, plays important roles in interacting with other proteins. For example, it contacts the elongation factor NusE, which is also the ribosomal S10 subunit. This interaction is essential for forming rRNA and λ gene antitermination complexes, as well as for the coupling of transcription and translation. Furthermore, the KOW contacts Rho for transcription termination (9, 11).
Fig. 1.
X-ray crystal structure of the P. furiosus Spt4/5. (A) Two molecules of Spt4/5 were present in the asymmetric unit. Spt4 and domains of Spt5 are denoted by a unique color and labeled. Zn2+ is depicted as a cyan sphere. One of the Spt4/5 heterodimers is partially transparent. Cα atoms of amino acid residues in Spt5 involved in heterodimer formation with Spt4 are shown as color spheres (green: NGN, blue: linker, orange: KOW), whereas those involved in contact with another Spt5 molecule found in the asymmetric unit are shown as gray spheres. (B) Amino acid sequence and structure alignments of archaeal and eukaryotic Spt5 and bacterial NusG. The amino acid residues corresponding to the NGN (green), linker (blue), and KOW (orange) are indicated by bars. Absolutely conserved residues are shown as white letters with red background, and highly conserved resides are indicated by red letters. Secondary structures of P. furiosus Spt5 (Pfu, determined in this work), S. cerevisiae Spt5 (Sce, PDB ID code 2EXU) (12), and E. coli NusG (9) (Eco, PDB ID code 2KO6 for NGN and PDB ID code 2JVV for KOW) are also shown. Amino acid residues making hydrophobic and basic patches on Spt5 for the coiled-coil and DNA bindings are indicated by yellow and blue dots. Sac, S. acidocaldarius; Mja, M. jannaschii; Has, H. sapiens; Dme, D. melanogaster; Bsu, B. subtilis; Tth, T. Thermophilus.
Eukaryotic Spt5 is the functional and structural counterpart of bacterial NusG. In addition to the NGN and KOW domains, eukaryotic Spt5 contains additional motifs including the N-terminal acidic region, four to five additional KOW motifs, and C-terminal repeats that are targets of regulatory kinases (12) (Fig. S1A). Eukaryotic Spt5 associates with Spt4 to form a Spt4/5 heterodimer, which is also called the DRB (5,6-dichloro-1-β-D-ribofuranosylbenzimidazole) sensitivity-inducing factor (DSIF) in higher eukaryotes (13). The Spt4/5 complex plays important roles in regulating transcription elongation both positively (14) and negatively (15).
Structural and biochemical studies of the archaeal RNAP transcription system revealed that it is an excellent model for dissecting the molecular basis of eukaryotic transcription, as well as providing unique data that may unify basic transcription mechanisms across all three domains of life (1618). The archaeal transcription apparatus, which includes RNAP and general transcription and elongation factors, is similar to the eukaryotic system. Archaeal Spt5 has only two domains, NGN and KOW, similar to the bacterial NusG, but it forms a heterodimer with Spt4 like the eukaryotic Spt4/5 complex (19) (Fig. S1). To understand how Spt4/5 regulates transcription elongation, we determined the crystal structure of archaeal Spt4/5 and the complex structure of RNAP-Spt4/5 using cryoelectron microscopy and single particle analysis.

Results and Discussion

X-ray Crystal Structure of Pyrococcus furiosus Spt4/5.

Crystal structures of archaeal Spt4/5 from Methanococcus jannaschii (19) and eukaryotic Spt4/5 from yeast (12) and human (20) revealed a highly conserved overall structure and architecture of the Spt4 and Spt5 dimerization surface. However, as these structures contain only the NGN of Spt5, the orientation of the KOW relative to NGN remains unknown. Here we have determined the crystal structure of the complete P. furiosus Spt4/5 at 1.8-Å resolution (Table S1). The P. furiosus Spt4 and Spt5 were produced independently in E. coli and purified as an Spt4/5 complex to homogeneity as determined by SDS-PAGE. The molecular weights of His6-tagged Spt4 and Spt5 are 11 and 17 kDa, respectively, and a native molecular mass of 25 kDa determined by size exclusion column chromatography indicates that Spt4/5 exist as a heterodimer in solution. The crystal structure of P. furiosus Spt4/5 was determined by molecular replacement, and refinement statistics are detailed in Table S1.
The overall structures of P. furiosus Spt4 and Spt5-NGN as well as the dimerization interface are similar to their homologues from M. jannaschii (19), yeast (12), and human (20) (Fig. 1 and Fig. S1). P. furiosus Spt4 consists of a NGN-binding domain (residues 30–61) and a zinc-binding domain (residues 2–29), with a zinc atom surrounded by four highly conserved cysteine residues (Fig. 1A). Spt5 consists of the NGN (residues 2–83) and KOW (residues 92–148) domains connected by an eight amino acid linker. There are two Spt4/5 heterodimers per asymmetric unit, and these structures are almost identical (0.257-Å root mean square deviations over 207 residues) and are related by a 2-fold noncrystallographic symmetry axis. The two heterodimers interact between NGN and KOW domains and also between KOWs (Fig. 1A).
We concluded that the Spt5-KOW is able to form multiple orientations relative to the Spt5-NGN in solution for the following reasons. First, about 78% of the total contact area (2,367 2) of Spt5 for Spt4 binding was found in the Spt5-NGN, and there is no interaction between NGN and KOW in the same Spt5 molecule. Several amino acid residues in the Spt5-KOW are involved in protein–protein contact with another Spt5 molecule in the asymmetric unit (contact area is 2,767 2, Fig. 1A). This crystallographic contact between two Spt5 molecules in the asymmetric unit may force orienting the KOW relative to the NGN. Accordingly, a solution structure of P. furiosus Spt4/5 in the complex with RNAP showed a different conformation of the NGN and KOW domains, as well as an extended linker compared with the crystal structure (described below).

Cryo-EM Reconstruction of the P. furiosus RNAP-Spt4/5 Complex.

Deletion mutagenesis studies of the bacterial and archaeal RNAPs have suggested that the RNAP clamp coiled-coil motif in the largest subunit, β′ in bacteria and RpoA1 in archaea, is a binding platform for NusG and Spt4/5 (19, 21). Nonetheless, the lack of structural models of the RNAP-NusG and RNAP-Spt4/5 complexes has hampered our understanding of how these factors influence RNAP transcription. To obtain molecular details of the interactions between RNAP and the elongation factor and structural insight into how the elongation factor influences RNAP transcription, we obtained the structure of P. furiosus RNAP in complex with Spt4/5 at 13-Å resolution using cryoelectron microscopy single particle analysis under a physiological condition (Figs. S2 and S3).
Eukaryotic Spt4/5 is only able to form a stable complex with the Pol II elongation complex with a nascent transcript longer than 18 nt (22, 23). In contrast, archaeal Spt4/5 is able to associate with RNAP independent of nucleic acids (19), and we took advantage of the simple and robust archaeal transcription system for obtaining homogeneous RNAP-Spt4/5 complexes suitable for cryo-EM studies.
The cryo-EM structure of the P. furiosus RNAP has been reported by two different groups (24, 25), one of which used an RNAP sample provided by us (24). In both cryo-EM maps, the P. furiosus RNAP resembles a “crab claw” with a protruding “stalk,” similar to the crystal structure of Sulfolobus sulfataricus RNAP (Fig. S4 A and B). There is a large cleft between the RNAP claw comprising the RpoA1 and RpoB subunits, which is proposed for binding the downstream and upstream double-stranded DNA.
The cryo-EM map of P. furiosus RNAP-Spt4/5 complex determined in this study also shows the characteristic overall shape of RNAP including the protruding stalk (Fig. 2A), allowing us to fit the crystal structure of S. sulfataricus RNAP (16) into the map unambiguously (Fig. 2B). In the cryo-EM reconstruction, however, a clear region of extra density lies across the cleft (labeled “a” in the panel). Instead of a clear cleft, the extra density bridges the RNAP claw, which together encircle a large channel. The extra density is positioned at the center of three RNAP domains called clamp, protrusion, and lobe and is weakly connected to another smaller extra density (labeled “b” in the panel) protruding from the large density (Fig. 2 B and C). We have assigned these two density regions to be Spt4/5. The Spt5-NGN has been assigned to be the RNAP binding domain (19); thereby we assigned density region a to be Spt4/Spt5-NGN and b to be the Spt5 KOW, respectively. To validate that density region a indeed corresponds to the Spt4/Spt5-NGN, not the Spt5-KOW domain, we carried out antibody labeling experiments against the 6xHis-tag at the N terminus of Spt4 as shown in Fig. 3. Briefly, we imaged the antibody labeled RNAP-Spt4/5 complex using negative stained EM. The labeled particles were picked and aligned to the cryo-EM reconstruction of the complex. Euler angles were assigned to these images by projection matching (Fig. 3 AD). Comparisons with the reprojections of the cryo-EM reconstructions along the same angles (Fig. 3 B and C) show a good correspondence between the particles and the reprojections. Additional density regions could be observed that have a characteristic Y shape of the IgGs (Fig. 3 C and D). The antibodies are clearly colocalized with the density a, which we assigned to be the Spt4/5-NGN domain (as indicated by magenta arrows), and have no overlap with density b (as indicated by orange arrows), which we have assigned to be the KOW domain.
Fig. 2.
Surface representations of the P. furiosus RNAP-Spt4/5 complex reconstruction. (A) Two different views of the RNAP-Spt4/5 complex reconstruction: from the top (looking from above the upstream DNA binding channel) and from the front (looking into the active site cleft, downstream and upstream DNAs are positioned on left and right, respectively). Positions of D/L and E/F subunits as well as RNAP domains (“prot”, protrusion) are indicated. (B) Partially transparent maps of A are shown with the S. sulfataricus RNAP crystal structure fitted into the cryo-EM maps. RpoA1 and RpoA2, dark gray; RpoB, light brown; other RNAP subunits, light gray. Positions of extra densities assigned for the Spt4/Spt5-NGN (a, green) and Spt5-KOW (b, orange) are indicated. (C) A magnified view of the interface between RNAP and Spt4/5. This view is the same as a boxed area in B. RNAP and Spt4/5 are shown as surface and cartoon models, respectively. The cryo-EM map is shown as mesh. Spt5-NGN, green; Spt5-linker, blue; Spt5-KOW, orange; Spt4, violet. (D) The Spt5-NGN and coiled-coil interaction. The cartoon models of Spt5-NGN (green), Spt5-linker (blue), and Spt4 (violet) are shown with partially transparent surface view. The RNAP coiled coil is shown as a gray tube. Hydrophobic residues of Spt5 involved in the coiled-coil interaction are depicted as sticks and colored yellow. Cα positions of the three hydrophobic residues (P261, L263, and I264) in the coiled coil are indicated as spheres.
Fig. 3.
IgG labeling of Spt4 in the RNAP-Spt4/5 complex. Monoclonal anti-poly-Histidine IgG labeled particles were aligned to the cryo-EM reconstruction of the complex and angles assigned by projection matching. (A) Three-dimensional surface rendering of cryoreconstruction viewed from the assigned angles. Purple arrows indicate the location of Spt4/5-NGN domain from the fitted model. Orange arrows indicate the location of the Spt5-KOW domain. (B) Reprojections from the 3D reconstruction along the assigned Euler angles. (C) Raw negatively stained immunocomplexes. White circles indicate the IgG density. Red outline shows the position of reprojected particle from B. (D) Same as C with arrows, showing the position of Spt4/5-NGN (magenta) and Spt5-KOW (orange). IgG density is also indicated (blue).
Next, we carried out rigid body fitting of the X-ray structure of P. furiosus Spt4/5 determined in this study (Fig. 2C), which showed good agreement between the crystal structure and the density shape. We also carried out energy minimization calculation for modeling the complex of RNAP coiled-coil motif and Spt4/5 (Fig. 2D). The hydrophobic patch of Spt5-NGN, including Y45 and F47, faces the tip of the coiled coil of the RpoA1 subunit as suggested by site-directed and deletion mutagenesis studies (19). In addition to the main interactions, our cryo-EM structure identified an additional previously undescribed contact site; α1 and α2 of Spt5-NGN fit into a cleft between the lobe and the protrusion (Fig. 2C). There is no interaction observed between RNAP and Spt4 in our model. This observation is in good agreement with biochemical data that the Spt5-NGN is the sole factor to form a complex with RNAP (19). The Spt5-KOW fits nicely into density b (Fig. 2C), outside the main RNAP density. To fit the Spt5-KOW into the cryo-EM map, the KOW was rotated about 90 ° toward the upstream DNA side starting from the crystal structure. No steric clash between Spt4 and the Spt5-KOW nor Spt4 and the Spt5-linker is observed for this KOW fitting. A binding site of bacterial transcription elongation factor RfaH, which is a paralog of E. coli NusG, was mapped on the coiled coil of RNAP, and the docking model of RNAP transcription elongation complex with RfaH was constructed (21). A location and orientation of RfaH relative to the coiled coil in the docking model are similar to those of the Spt5-NGN in the cryo-EM RNAP-Spt4/5 complex structure.

Modeling of the Transcription Elongation Complex with Spt4/5.

The DNA binding channel of multisubunit RNAP is positioned between the RNAP claw, and the space of the channel is modulated by the position of the RNAP clamp. The crystal structures of transcription elongation complexes consisting of RNAP and the DNA/RNA hybrid, from T. thermophilus (26) and Pol II (27), showed that the clamp is in the closed position in the transcribing RNAP, but the channel is still partially open, perhaps explaining the property for transcription to pause in the absence of elongation factors. The cryo-EM reconstruction of the RNAP-Spt4/5 complex in this study revealed that Spt4/5 binds between the RNAP claw, thereby completely encircling the DNA binding channel. This closure would prevent DNA from disengaging from the transcribing RNAP and may provide an explanation as to why Spt4/5 is able to enhance transcription processivity. Complete closure of the DNA channel is reminiscent of the DNA sliding clamp in the DNA replication fork, the β-clamp in bacteria, and PCNA in eukaryotes, which enhances the processivity of DNA polymerase (28). The enclosure of DNA by proteins is also found in many processive ring helicases such as RuvB, E1, Large T-antigen, and MCM (minichromosome maintenance) (29).
In the cryo-EM reconstruction of the RNAP-Spt4/5 complex, the diameter of the channel for the downstream DNA is much wider than that for upstream DNA (Fig. 2A). To obtain further structural insight into how the transcription bubble is accommodated in the RNAP-Spt4/5 complex and also how Spt4/5 influences RNAP transcription, the DNA/RNA scaffold was modeled into the RNAP-Spt4/5 complex. Although many crystal structures of eukaryotic and bacterial elongation complexes have been determined, none of them shows a clear location for the nontemplate DNA in the transcription bubble nor the upstream double-stranded DNA. Recent single-molecule FRET experiments constructed the complete elongation complex model of yeast Pol II (30), and we have used this DNA/RNA scaffold for modeling the archaeal elongation complex. The yeast elongation complex model is overlaid with the archaeal RNAP using secondary structure matching. The downstream DNA fits perfectly into the DNA binding channel whereas the upstream DNA had to be tilted ∼30° toward the RNAP wall to avoid steric clash with the density corresponding to Spt4/5 (Fig. 4A). It is important to note that the position of the upstream DNA has not been determined structurally. Only a small region of the upstream DNA is protected by RNAP in the DNA footprinting experiment, and a limited amount of DNA cross-linking was observed in this area (31). The upstream DNA in the elongation complex is therefore intrinsically flexible and may occupy multiple positions during transcription elongation. Spt4/5 in the elongation complex is ideally positioned to function as an “upstream DNA holder” and may be able to hold the upstream DNA in a confined location.
Fig. 4.
Model of the P. furiosus RNAP transcription elongation complex with Spt4/5. (A) Two different views—front and side—of the RNAP cryo-EM reconstruction with the DNA/RNA model (template DNA, cyan; nontemplate DNA, black; RNA, red). Positions of D/L and E/F subunits, RNAP domains and Spt4/5 domains (Spt4/Spt5-NGN, NGN; Spt5-KOW, KOW) are indicated. Locations of downstream (d-DNA) and upstream (u-DNA) double-stranded DNAs are indicated. A map of the side view was sliced to show a transcription bubble and the active site (AC) inside the DNA binding channel. (B) The interaction between Spt4/5 and nucleic acid in the transcribing RNAP. A magnified view showing only DNA/RNA, Spt4/Spt5-NGN, and coiled coil (cc) of clamp. An orientation of this view is the same as A, Right. The surface of Spt4/5 is color coded according to electrostatic surface potential (negative, red; neutral, white; positive, blue).
A basic region of Spt5-NGN is located near the nontemplate DNA within the transcription bubble (Fig. 4B). In this basic patch, R9, K43, and R67 are highly conserved in archaea, but R61 and R64 are less conserved (Fig. 1B). Some of these residues are conserved in eukaryotic Spt5 and bacterial NusG, but interestingly, each residue is conserved only in either eukaryote or bacteria; e.g., R9/K43/R67 and R61 are conserved in only eukaryote and bacteria, respectively. The different amino acids in this region may explain their unique functions only found in particular domains of life. The close proximity of the Spt5-NGN and the nontemplate DNA within the transcription bubble in the model is able to explain the result whereby B. subtilis NusG contacts the nontemplate DNA for stabilizing transcriptional pausing (7).
In addition to the interaction with proteins, the KOW motif has been shown to bind DNA and RNA (32). In the model of the transcription elongation complex with Spt4/5 (Fig. 4A), the Spt5-KOW locates near the upstream DNA, which allows it to contact DNA. A recent photo-cross-linking experiment with the Drosophila Pol II transcription elongation complex in the presence of Spt4/5 has shown that the Spt5 subunit contacts a nascent transcript if the RNA length is longer than 18 nt (22). In our model (Fig. 4A), the Spt5-KOW locates far from the 16 nt RNA, which just begins to emerge from the surface of RNAP suggesting that eukaryotic Spt5 uses its second to fifth KOW motifs but not the first KOW motif (Fig. S1A) for contacting nascent RNA in the transcription elongation complex.

Concluding Remarks

A recent single-molecule study of E. coli RNAP transcription revealed that NusG enhances the processivity of transcription by increasing the pause-free speed of RNAP elongation and suppressing the entry into long-lifetime pauses (6). In this study, the cryo-EM structure of the RNAP-Spt4/5 complex revealed that the elongation factor encircles the DNA, similar to the DNA clamp in DNA polymerase, to promote processivity. Furthermore, Spt4/5 is able to contact two different positions of DNA in the transcribing RNAP that include the upstream DNA and the single-stranded nontemplate DNA (Fig. 4). The restriction of the upstream DNA by Spt4/5 may facilitate (i) ejecting the transcribed DNA template from the DNA channel and (ii) reannealing between the template and nontemplate DNA strands at the upstream end of the transcription bubble (Fig. 4), promoting the RNAP toward its posttranslocated state. The interactions between the nontemplate DNA in the transcription bubble and the basic patch of Spt5-NGN could prevent DNA secondary structure formation in the transcription bubble. Combinations of these effects by the transcription elongation factor would prevent elemental and backtracked pauses of RNAP transcription thereby improving the processivity of transcription (1, 6). The interesting remaining questions are which interaction affects what aspect of transcription and whether one interaction influences only one set of parameters? The structure of archaeal RNAP-Spt4/5 complex presented in this study provides a structural framework for further biochemical and biophysical studies to answer these questions.

Materials and Methods

Protein Preparations and X-Ray Crystal Structure Determination.

(i) Cloning of P. furiosus Spt4 and Spt5 genes, (ii) Spt4 and Spt5 expressions and purification of Spt4/5 complex, (iii) crystallization and determination of the X-ray crystal structure of P. furiosus Spt4/5, and (iv) P. furiosus RNAP purification and the RNAP-Spt4/5 complex formation are described in SI Text.

Cryo-Electron Microscopy.

P. furiosus RNAP-Spt4/5 complex (2 μL of ∼0.1 mg/mL) was applied to glow-discharged 300 mesh holey carbon grids (Quantifoil). Excess sample was blotted and flash frozen in liquid ethane cooled by liquid nitrogen (-183 °C) to form vitreous ice. Grids were prepared under controlled environmental conditions (100% humidity, 4 °C) using a vitrobot (FEI). Data were collected at 50,000× magnification using a Philips CM200 field emission gun (FEG) electron microscope operating at 200 kV under low-dose conditions (10 e-/2), and over a range of nominal defoci (-0.5 μm to -3.5 μm). Images were collected directly on a 4k × 4k CCD camera (F415 from Tietz Video and Imaging Processing GmbH), giving a pixel size of 1.76 Å (Fig. S2). (i) Image processing, (ii) IgG labeling of Spt4 in the RNAP-Spt4/5 complex, and (iii) fitting of the S. sulfataricus RNAP and the P. furiosus Spt4/5 X-ray crystal structures are described in SI Text.

Data Availability

Data deposition: Coordinate and structure factor have been deposited in the Protein Data Bank, www.pdb.org (PDB ID code 3P8B). The cryo-EM map corresponding to the RNAP-Spt4/5 complex has been deposited in the EMDataBank, www.emdatabank.org (EMD ID code 1840).


We thank the staff at F1 of the MacCEHSS (Macromolecular Diffraction at Cornell High Energy Synchrotron Source) for support crystallographic data collection. We thank P. Babitzke, D. S. Gilmour, and L. B. Rothman-Denes for critical readings of the manuscript. We thank I. Artsimovitch for sending a coordinate of the Tth TEC-RhaH complex model. Figures were prepared using PyMOL (http://pymol.sourceforge.net/) and University of California, San Francisco (UCSF) Chimera (33). This work was supported by National Institutes of Health (NIH) Grant GM087350 (to K.S.M.) and Wellcome Trust (to X.Z). Center for Hybrid and Embedded Software Systems is supported by the National Science Foundation (NSF) and NIH/National Institute of General Medical Sciences via NSF Award DMR-0225180, and the MacCHESS resource is supported by NIH/National Center for Research Resources Award RR-01646.

Supporting Information

Supporting Information (PDF)
Supporting Information


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Information & Authors


Published in

Go to Proceedings of the National Academy of Sciences
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Proceedings of the National Academy of Sciences
Vol. 108 | No. 2
January 11, 2011
PubMed: 21187417


Data Availability

Data deposition: Coordinate and structure factor have been deposited in the Protein Data Bank, www.pdb.org (PDB ID code 3P8B). The cryo-EM map corresponding to the RNAP-Spt4/5 complex has been deposited in the EMDataBank, www.emdatabank.org (EMD ID code 1840).

Submission history

Published online: December 27, 2010
Published in issue: January 11, 2011


  1. cryo-EM
  2. Spt4/5-DSIF-NusG
  3. X-ray crystallography


We thank the staff at F1 of the MacCEHSS (Macromolecular Diffraction at Cornell High Energy Synchrotron Source) for support crystallographic data collection. We thank P. Babitzke, D. S. Gilmour, and L. B. Rothman-Denes for critical readings of the manuscript. We thank I. Artsimovitch for sending a coordinate of the Tth TEC-RhaH complex model. Figures were prepared using PyMOL (http://pymol.sourceforge.net/) and University of California, San Francisco (UCSF) Chimera (33). This work was supported by National Institutes of Health (NIH) Grant GM087350 (to K.S.M.) and Wellcome Trust (to X.Z). Center for Hybrid and Embedded Software Systems is supported by the National Science Foundation (NSF) and NIH/National Institute of General Medical Sciences via NSF Award DMR-0225180, and the MacCHESS resource is supported by NIH/National Center for Research Resources Award RR-01646.


*This Direct Submission article had a prearranged editor.



Brianna J. Klein1
Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, PA 16802; and
Daniel Bose1
Division of Molecular Biosciences, Centre for Structural Biology, Department of Life Sciences, Faculty of Natural Sciences, Imperial College London, London SW7 2AZ, United Kingdom
Kevin J. Baker
Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, PA 16802; and
Zahirah M. Yusoff
Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, PA 16802; and
Xiaodong Zhang2 [email protected]
Division of Molecular Biosciences, Centre for Structural Biology, Department of Life Sciences, Faculty of Natural Sciences, Imperial College London, London SW7 2AZ, United Kingdom
Katsuhiko S. Murakami2 [email protected]
Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, PA 16802; and


To whom correspondence may be addressed. E-mail: [email protected] or [email protected].
Author contributions: X.Z. and K.S.M. designed research; B.J.K., D.B., and Z.M.Y. performed research; B.J.K., D.B., X.Z., and K.S.M. analyzed data; K.J.B. contributed new reagents/analytic tools; and X.Z. and K.S.M. wrote the paper.
B.J.K. and D.B. contributed equally to this work.

Competing Interests

The authors declare no conflict of interest.

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    RNA polymerase and transcription elongation factor Spt4/5 complex structure
    Proceedings of the National Academy of Sciences
    • Vol. 108
    • No. 2
    • pp. 435-887







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