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. 2011 Jan 11;108(2):546-50.
doi: 10.1073/pnas.1013828108. Epub 2010 Dec 27.

RNA polymerase and transcription elongation factor Spt4/5 complex structure

Brianna J Klein  1 , Daniel Bose, Kevin J Baker, Zahirah M Yusoff, Xiaodong Zhang, Katsuhiko S Murakami
Affiliations

RNA polymerase and transcription elongation factor Spt4/5 complex structure

Brianna J Klein et al. Proc Natl Acad Sci U S A. .

Abstract

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.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
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.
Fig. 2.
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.
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).
Fig. 4.
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).
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