Crystal structure of Pfu RNAP clamp–Spt4/5 complex. (A) Schematic of domain architecture of Spt4/5 and NusG in the three kingdoms of life. Zn, zinc-binding motif; NGN, NusG N-terminal domain; KOW, Kyrides–Onzonis–Woese domain; CTR, C-terminal repeat region. (B) Ribbon model of the Pfu RNAP clamp–Spt4/5 complex crystal structure. The views correspond approximately to the front and side views of RNAP II used before (Kettenberger et al, 2004) and are related by a 90° rotation around a vertical axis. Spt4, the Spt5 NGN domain and the RNAP clamp are in yellow, green, and red, respectively, and these colours are used throughout. The clamp secondary structure elements are numbered in accordance with the RNAP II structure. (C) Amino-acid sequence alignments of Spt4, the Spt5 NGN domain, and the clamp coiled coil. Sequences from the archaea P. furiosus (Pfu), S. solfataricus (Sso), M. janaschii (Mja), the eukarya S. cerevisiae (Sce) and H. sapiens (Hsa), and the bacteria T. thermophilus (Tth) and E. coli (Eco) were used. Secondary structure elements are indicated as arrows (β-strands) or rods (α-helices). Loops are indicated with solid lines. Hydrophobic and polar residues that are part of the Spt5 NGN–clamp coiled-coil interface are marked with black dots and triangles, respectively. Conserved and positively charged surface residues on Spt4/5 are marked with black squares.

Models of RNAP complexes with Spt4/5 or NusG. Ribbon models for the eukaryotic RNAP II–Spt4/5 complex (left), the archaeal RNAP–Spt4/5 (middle), and the bacterial RNAP–NusG complex (right). For each model, the front (top) and side views (bottom) are shown that are also used in Figure 2. Key elements of RNAP are labelled, including a non-conserved domain (NCD) in the largest bacterial RNAP subunit (omitted from the front view). Compare text for details on model generation.

Relative positions of elements important for additional Spt5/NusG functions. The complete yeast RNAP II elongation complex with bound Spt4/5 (shown in Figure 5) is viewed from the back (related to the front view of Figures 2, 4, and 5 by a 180° rotation around a vertical axis). The KOW1 domain of Spt5 was positioned onto the model by superposing a representative NusG crystal structure (PDB code 1NPP) via its NGN domain and thus positioning its KOW domain relative to the Spt5 NGN. Important RNAP II elements are labelled. Exiting RNA, the C-terminal KOW domains and C-terminal repeat region of Spt5 (CTR), and the RNAP II largest subunit C-terminal domain (CTD) extend towards the same side around the Rpb4/7 subcomplex. For details compare text.

Spt4/5 binds endogenous RNAPs and a recombinant RNAP clamp. (A) Yeast (S. cerevisiae) complexes. SDS–PAGE analysis (Coomassie staining) of purified recombinant Spt4/5 (containing full-length Spt4 and Spt5 residues 283–853), endogenous RNAP II, and the RNAP II–Spt4/5 complex after size exclusion chromatography. The identity of the bands was confirmed by mass spectrometry. (B) Archaeal (P. furiosus, Pfu) complexes. SDS–PAGE analysis (Coomassie staining) of purified recombinant Spt4/5 (containing full-length Spt4 and Spt5), endogenous RNAP, and the RNAP–Spt4/5 complex after size exclusion chromatography. (C) Schematic of the fusion protein used to prepare the recombinant Pfu clamp domain. Polypeptide regions of the three largest RNAP subunits B, A′, and A′′ were fused by two short linkers. Bordering residue numbers are indicated. (D) SDS–PAGE analysis (Coomassie staining) of recombinant Pfu clamp, Spt4/5, and clamp–Spt4/5 complexes.

The Spt5 NGN–RNAP clamp coiled-coil interface. Zoom-in view of the structure in Figure 2 showing the Spt5 NGN domain (green) and the coiled coil of the RNAP clamp (red) as ribbon models. Residues that form the Spt5–clamp interface are depicted as stick models and labelled. Hydrogen bonds and salt bridges are shown as black and red dashed lines, respectively. The views correspond approximately to the front and side views used in Figure 2, albeit rotated slightly around a vertical axis to allow for optimal viewing of the interface.

Model of the complete yeast RNAP II–Spt4/5 elongation complex. (A) Model of the complete yeast RNAP II elongation complex with bound Spt4/5. Proteins are shown as molecular surfaces with key domains highlighted in colour and labelled. Nucleic acids are shown as ribbon models with the DNA template, DNA non-template, and the RNA in blue, cyan, and red, respectively. The views are as in Figures 2 and 4. For details on how the model was prepared please compare text. (B) The model shown in (A) but with the RNAP II surface in silver and the Spt4/5 solvent-accessible surface coloured according to electrostatic charge distribution (blue, positive charge; red, negative charge) as calculated by APBS (Baker et al, 2001).