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Architecture of the RNA polymerase-Spt4/5 complex and basis of universal transcription processivity.

Martinez-Rucobo FW, Sainsbury S, Cheung AC, Cramer P - EMBO J. (2011)

Bottom Line: The structure revealed a conserved Spt5-RNAP interface and enabled modelling of complexes of Spt4/5 counterparts with RNAPs from all kingdoms of life, and of the complete yeast RNAP II elongation complex with bound Spt4/5.The N-terminal NGN domain of Spt5/NusG closes the RNAP active centre cleft to lock nucleic acids and render the elongation complex stable and processive.The C-terminal KOW1 domain is mobile, but its location is restricted to a region between the RNAP clamp and wall above the RNA exit tunnel, where it may interact with RNA and/or other factors.

View Article: PubMed Central - PubMed

Affiliation: Gene Center and Department of Biochemistry, Center for Integrated Protein Science Munich (CIPSM), Ludwig-Maximilians-Universität München, Munich, Germany.

ABSTRACT
Related RNA polymerases (RNAPs) carry out cellular gene transcription in all three kingdoms of life. The universal conservation of the transcription machinery extends to a single RNAP-associated factor, Spt5 (or NusG in bacteria), which renders RNAP processive and may have arisen early to permit evolution of long genes. Spt5 associates with Spt4 to form the Spt4/5 heterodimer. Here, we present the crystal structure of archaeal Spt4/5 bound to the RNAP clamp domain, which forms one side of the RNAP active centre cleft. The structure revealed a conserved Spt5-RNAP interface and enabled modelling of complexes of Spt4/5 counterparts with RNAPs from all kingdoms of life, and of the complete yeast RNAP II elongation complex with bound Spt4/5. The N-terminal NGN domain of Spt5/NusG closes the RNAP active centre cleft to lock nucleic acids and render the elongation complex stable and processive. The C-terminal KOW1 domain is mobile, but its location is restricted to a region between the RNAP clamp and wall above the RNA exit tunnel, where it may interact with RNA and/or other factors.

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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.
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f2: 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.

Mentions: The purified rClamp–Spt4/5 complex could be crystallized and its X-ray structure determined at 3.3 Å resolution (Materials and methods). For structure solution, we combined experimental phases obtained from anomalous diffraction of four zinc ions (three in the clamp and one in Spt4) with model phases obtained by molecular replacement with the S. cerevisiae clamp structure (Armache et al, 2005). The Methanococcus janaschii Spt4/5 structure (Hirtreiter et al, 2010) was then fitted into the experimentally phased electron density map alongside the clamp structure, and after repeated cycles of rebuilding and refinement, an atomic model of the complex was refined that only lacked the Spt5 KOW domain, which was disordered (Table I; Figure 2). In the rClamp–Spt4/5 complex, the structures of free Spt4/5 and the clamp in free RNAP are essentially unaltered, except for minor local conformational changes.


Architecture of the RNA polymerase-Spt4/5 complex and basis of universal transcription processivity.

Martinez-Rucobo FW, Sainsbury S, Cheung AC, Cramer P - EMBO J. (2011)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3094117&req=5

f2: 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.
Mentions: The purified rClamp–Spt4/5 complex could be crystallized and its X-ray structure determined at 3.3 Å resolution (Materials and methods). For structure solution, we combined experimental phases obtained from anomalous diffraction of four zinc ions (three in the clamp and one in Spt4) with model phases obtained by molecular replacement with the S. cerevisiae clamp structure (Armache et al, 2005). The Methanococcus janaschii Spt4/5 structure (Hirtreiter et al, 2010) was then fitted into the experimentally phased electron density map alongside the clamp structure, and after repeated cycles of rebuilding and refinement, an atomic model of the complex was refined that only lacked the Spt5 KOW domain, which was disordered (Table I; Figure 2). In the rClamp–Spt4/5 complex, the structures of free Spt4/5 and the clamp in free RNAP are essentially unaltered, except for minor local conformational changes.

Bottom Line: The structure revealed a conserved Spt5-RNAP interface and enabled modelling of complexes of Spt4/5 counterparts with RNAPs from all kingdoms of life, and of the complete yeast RNAP II elongation complex with bound Spt4/5.The N-terminal NGN domain of Spt5/NusG closes the RNAP active centre cleft to lock nucleic acids and render the elongation complex stable and processive.The C-terminal KOW1 domain is mobile, but its location is restricted to a region between the RNAP clamp and wall above the RNA exit tunnel, where it may interact with RNA and/or other factors.

View Article: PubMed Central - PubMed

Affiliation: Gene Center and Department of Biochemistry, Center for Integrated Protein Science Munich (CIPSM), Ludwig-Maximilians-Universität München, Munich, Germany.

ABSTRACT
Related RNA polymerases (RNAPs) carry out cellular gene transcription in all three kingdoms of life. The universal conservation of the transcription machinery extends to a single RNAP-associated factor, Spt5 (or NusG in bacteria), which renders RNAP processive and may have arisen early to permit evolution of long genes. Spt5 associates with Spt4 to form the Spt4/5 heterodimer. Here, we present the crystal structure of archaeal Spt4/5 bound to the RNAP clamp domain, which forms one side of the RNAP active centre cleft. The structure revealed a conserved Spt5-RNAP interface and enabled modelling of complexes of Spt4/5 counterparts with RNAPs from all kingdoms of life, and of the complete yeast RNAP II elongation complex with bound Spt4/5. The N-terminal NGN domain of Spt5/NusG closes the RNAP active centre cleft to lock nucleic acids and render the elongation complex stable and processive. The C-terminal KOW1 domain is mobile, but its location is restricted to a region between the RNAP clamp and wall above the RNA exit tunnel, where it may interact with RNA and/or other factors.

Show MeSH
Related in: MedlinePlus