The Structure of a Transcribing T7 RNA Polymerase in Transition from Initiation to Elongation

Department of Molecular Biophysics and Biochemistry, Yale University, 266 Whitney Avenue, New Haven, CT 06520-8114, USA.
Science (Impact Factor: 33.61). 11/2008; 322(5901):553-7. DOI: 10.1126/science.1163433
Source: PubMed


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.

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    • "In the structural transition from IC3 to IC7, promoter, the promoter binding domain and the specificity loop move as a rigid body away from the C-terminal domain, presumably pushed by the hybrid (Durniak K J, et al. 2008). Consequently, enzyme-promoter interactions are virtually unchanged, and space opens up to accommodate more template DNA and newly synthesized RNA within the enzyme. "
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    ABSTRACT: During transcription initiation, RNA polymerase binds tightly to the promoter DNA defining the start of transcription, transcribes comparatively slowly, and frequently releases short transcripts (3-8 nucleotides) in a process called abortive cycling. Transitioning to elongation, the second phase of transcription, the polymerase dissociates from the promoter while RNA synthesis continues. Elongation is characterized by higher rates of transcription and tight binding to the RNA transcript. The RNA polymerase from enterophage T7 (T7 RNAP) has been used as a model to understand the mechanism of transcription in general, and the transition from initiation to elongation specifically. This single-subunit enzyme undergoes dramatic conformational changes during this transition to support the changing requirements of nucleic acid interactions while continuously maintaining polymerase function. Crystal structures, available of multiple stages of the initiation complex and of the elongation complex, combined with biochemical and biophysical data, offer molecular detail of the transition. Some of the crystal structures contain a variant of T7 RNAP where proline 266 is substituted by leucine. This variant shows less abortive products and altered timing of transition, and is a valuable tool to study these processes. The structural transitions from early to late initiation are well understood and are consistent with solution data. The timing of events and the structural intermediates in the transition from late initiation to elongation are less well understood, but the available data allows one to formulate testable models of the transition to guide further research.
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    • "Distribution of that kind could play a crucial role in the proper orientation of the protein towards the DNA during initial steps of promoter recognition: non-specific binding and promoter location. It is known (Cheetham, Jeruzalmi, & Steitz, 1999, Durniak, Bailey & Steitz 2008) that there are two domains responsible for promoter binding during transcription initiation step: promoter-binding domain (PBD) formed by six a-helical bundle (residues 72-150 and 191-267) and specificity loop (residues 739-770). Two parts of PDB are enclosed specificity loop constituting monolithic promoter recognition domain (Fig. 3A). "
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    ABSTRACT: The entire T7 bacteriophage genome contains 39937 base pairs (Database NCBI RefSeq N1001604). Here, electrostatic potential distribution around double helical T7 DNA was calculated by Coulomb method using the computer program of Sorokin A.A. ([email protected] /* */). Electrostatic profiles of 17 promoters recognized by T7 phage-specific RNA polymerase were analyzed. It was shown that electrostatic profiles of all T7 RNA polymerase-specific promoters can be characterized by distinctive motifs which are specific for each promoter class. Comparative analysis of electrostatic profiles of native T7 promoters of different classes demonstrates that T7 RNA polymerase can differentiate them due to their electrostatic features.
    Full-text · Article · Jul 2013 · Journal of biomolecular Structure & Dynamics
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    • "Synthesis of the first 8 nt does not involve major conformational changes, the second stage occurring between synthesis of 9 and 14 nt includes a major conformational change of the T7-RNAP observed in processive ECs (51). Analysis of T7-RNAP bound to promoter DNA containing an 8-nt RNA revealed rotation of the promoter bound domain in the N-terminus and the bound promoter by ∼45°, allowing the active site to accommodate a growing heteroduplex as required in processive elongation complexes (52). It is tempting to speculate that the major transition involving repositioning of subunit H in the archaeal enzyme and establishing the processive conformation of the enzyme encountered in ECs occurs also during the transition from the first to the second stage in archaea. "
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    ABSTRACT: The lower jaws of archaeal RNA polymerase and eukaryotic RNA polymerase II include orthologous subunits H and Rpb5, respectively. The tertiary structure of H is very similar to the structure of the C-terminal domain of Rpb5, and both subunits are proximal to downstream DNA in pre-initiation complexes. Analyses of reconstituted euryarchaeal polymerase lacking subunit H revealed that H is important for open complex formation and initial transcription. Eukaryotic Rpb5 rescues activity of the ΔH enzyme indicating a strong conservation of function for this subunit from archaea to eukaryotes. Photochemical cross-linking in elongation complexes revealed a striking structural rearrangement of RNA polymerase, bringing subunit H near the transcribed DNA strand one helical turn downstream of the active center, in contrast to the positioning observed in preinitiation complexes. The rearrangement of subunits H and A′′ suggest a major conformational change in the archaeal RNAP lower jaw upon formation of the elongation complex.
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