PDBsum entry 3e2e

Transferase/RNA/DNA

PDB id: 3e2e

Contents

Protein chain

832 a.a.

DNA/RNA

Waters ×73

References listed in PDB file

Key reference

Title

The structure of a transcribing t7 RNA polymerase in transition from initiation to elongation.

Authors

K.J.Durniak, S.Bailey, T.A.Steitz.

Ref.

Science, 2008, 322, 553-557. [DOI no: 10.1126/science.1163433]

PubMed id

18948533

Abstract

Structural studies of the T7 bacteriophage DNA-dependent RNA polymerase (T7 RNAP) have shown that the conformation of the amino-terminal domain changes substantially between the initiation and elongation phases of transcription, but how this transition is achieved remains unclear. We report crystal structures of T7 RNAP bound to promoter DNA containing either a 7- or an 8-nucleotide (nt) RNA transcript that illuminate intermediate states along the transition pathway. The amino-terminal domain comprises the C-helix subdomain and the promoter binding domain (PBD), which consists of two segments separated by subdomain H. The structures of the intermediate complex reveal that the PBD and the bound promoter rotate by approximately 45 degrees upon synthesis of an 8-nt RNA transcript. This allows the promoter contacts to be maintained while the active site is expanded to accommodate a growing heteroduplex. The C-helix subdomain moves modestly toward its elongation conformation, whereas subdomain H remains in its initiation- rather than its elongation-phase location, more than 70 angstroms away.

Figure 1.
Fig. 1. A Comparison of the structures of T7 RNAP initiation, intermediate, and elongation complexes. The molecules have been similarly oriented by superposition of their palm domains. The C-terminal domain is shown as a surface with the thumb domain removed to allow views of the DNA and RNA. The nontemplate strand is shown in light green, the template in blue, and the RNA transcript in red. Subdomains of the N-terminal domain are colored yellow (C-helix), green (subdomain H), and purple (PBD). (A) The initiation complex bound to the promoter has a 3-nt transcript with its 5' end in contact with the PBD. (B) The elongation complex exhibits a 220° right-hand rotation of the PBD, a refolding of subdomain H onto the top of the polymerase, and the formation of an elongated C-helix subdomain, when compared to the initiation complex. (C) (Top) The T7 RNAP in the 7-nt RNA intermediate complex is bound to both promoter and downstream DNA. The PBD has rotated by 40° away from the C-terminal domain, avoiding a steric clash with the transcript and allowing for 7 bp of heteroduplex to form in the active site. (Bottom) A schematic drawing of the sequences constituting the subdomains of the N-terminal domain.

Figure 5.
Fig. 5. Arrangement of the downstream DNA. (A) The downstream duplex is rotated by 30° toward the N-terminal domain compared to its position in the elongation complex (shown in gray). (B) The angle between the upstream and downstream duplex DNA is about 40°, bringing the phosphate backbones within 6 Å of each other. (C) The refolded subdomain H from the structure of the elongation complex (gray) creates a clash with the position of the downstream DNA as observed in the intermediate complex. Residues involved in the clash are shown as spheres. (D) A close-up view of the 5' end of the 7-nt RNA reveals that a modeled extension of the RNA by three additional nucleotides would clash with the specificity loop in the position observed in the intermediate complex.

The above figures are reprinted by permission from the AAAs: Science (2008, 322, 553-557) copyright 2008.