Structural studies of the T7 bacteriophage DNA-dependent RNA polymerase (T7 RNAP) have shown large structural differences between its initiation and elongation phases of transcription, but the mechanism by which this transition is achieved remains unclear. Crystal structures of T7 RNAP bound to promoter DNA containing either a seven or an eight nucleotide (nt) RNA transcript, now illuminate intermediate states along the transition pathway. These intermediate complex structures reveal that the six helix promoter-binding subdomain and the bound promoter rotate by approximately 45° upon synthesis of an 8nt RNA transcript, thereby allowing the maintenance of promoter contacts while expanding the active site to accommodate a growing heteroduplex. Although modest movements in the C-helix subdomain towards its elongation conformation are also observed, subdomain H undergoes little change.
RNA polymerases (RNAPs) exhibit three phases of transcription – initiation, elongation and termination. The initiation and elongation phases, have been studied extensively in the T7 RNAP system by biochemical and structural approaches [reviewed in (1, 2)]. During the initiation phase, the RNAP binds to a specific promoter DNA sequence, opens the DNA duplex and feeds the template strand into the active site (3, 4). The structure of a T7 RNAP initiation complex identified a six-helix bundle sub-domain (residues 72-150, 191-267), here termed the promoter binding domain (PBD), that is responsible for many of the interactions with the 17 base-pair (bp) promoter and, in part, for melting the DNA duplex (3, 4) (Fig 1A). While remaining bound to the promoter, the polymerase produces many short transcripts from 2-12 nucleotides (nt) in length (5, 6), a process often referred to as abortive synthesis. After the transition to the elongation phase and release of the promoter, the polymerase proceeds processively down the DNA template producing a full length RNA transcript. Comparison of the structures of the T7 RNAP initiation and elongation complexes revealed extensive conformational changes within the N-terminal 267 residues (N-terminal domain) and little change in the rest of the RNAP (Fig 1B) (7, 8). A rigid body rotation of the PBD as well as the refolding of the N-terminal C-helix (residues 28-71) and H (residues 151-190) subdomains are responsible for abolishing the promoter binding site, enlarging the active site and creating an exit tunnel for the RNA transcript.
The structural changes within the N-terminal domain account for the increased stability and the processivity of the elongation complex, yet provide little insight into how the polymerase is able to accommodate a growing transcript while maintaining its promoter contacts during the abortive synthesis phase. The structure of the initiation complex contains a 3nt RNA transcript in the active site DNA:RNA heteroduplex. Modeling the elongation of the RNA transcript by the addition of a single RNA nucleotide produced a steric clash with the PBD and incorporation of an additional NTP destroyed the crystal (4). Covalent cross-linking of the template strand to the RNA transcript established that the heteroduplex can be as long as 8bp (9, 10) thereby eliminating the possibility that the heteroduplex might be only 3bp long (4). The enlarged active site observed in the structure of the elongation complex (Fig 1B) can accommodate a 7-8bp heteroduplex, however the new orientation of the PBD abolishes the promoter binding site. Since biochemical data suggest that the polymerase remains bound to the promoter with transcripts as long as 8 to 10nt, the question of how the polymerase accommodates a growing RNA transcript while maintaining its promoter contacts during the transition from initiation to elongation is raised.
Cross-linking, mutagenesis and proteolytic digestion experiments have provided information relating the length of the RNA transcript to changes within the N-terminal domain (11-13), and suggest the existence of at least one intermediate structure. A common conclusion from the biochemical data is that the transition mechanism consists of two stages. Cross-linking and mutagenesis experiments that prevent major refolding of the T7 RNAP structure (11-15) suggest that the first stage allows synthesis of up to 8nt of RNA with minimal changes to the N-terminal sub-domains. The second stage is presumed to include the major refolding events that occur during the synthesis of 9-14nt and allow the stable elongation complex to form (16). A transition mechanism consisting of two stages has also been proposed for the larger, multi-subunit eukaryotic RNAPs (17).
Models of the transition from initiation to elongation proposed previously have suggested that the N-terminal domain undergoes a gradual structural rearrangement in order to accommodate an 8bp heteroduplex(7, 8). One model proposes a 10Å translation of the PBD and subdomain H away from the active site (18) , while another suggests the PBD can maintain its promoter contacts after rotating into the position observed in the elongation complex (7). None of these models are entirely consistent with the biochemical data (13, 19). However, while this manuscript was in preparation, Tang et al. posited that a rotation of the N-terminal domain could accommodate their FRET data as well as previous biochemical data (2). Although these new FRET data predict a 20° rotation, they do not provide the direction of the rotation or which components are rotating.
The structures of T7 RNAP bound to promoter DNA containing either a 7nt or 8nt RNA transcript presented here represent complexes that are between the early initiation and the elongation phases. These structures exhibit the domain movements that are necessary to accommodate transcript elongation during the abortive synthesis phase, and provide a model that is consistent with all of the biochemical data as well as the two-stage transition mechanism.