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: 31.48). 11/2008; 322(5901):553-7. DOI: 10.1126/science.1163433
Source: PubMed

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.

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    ABSTRACT: Transcription through chromatin by different RNA polymerases produces different biological outcomes and is accompanied by either nucleosome survival at the original location (Pol II-type mechanism) or backward nucleosome translocation along DNA (Pol III-type mechanism). It has been proposed that differences in the structure of the key intermediates formed during transcription dictate the fate of the nucleosomes. To evaluate this possibility, structure of the key intermediate formed during transcription by Pol III-type mechanism was studied by DNase I footprinting and molecular modeling. The Pol III-type mechanism is characterized by less efficient formation of the key intermediate required for nucleosome survival (Ø-loop, Pol II-type mechanism), most likely due to steric interference between the RNA polymerase and DNA in the Ø-loop. The data suggest that the lower efficiency of Ø-loop formation induces formation of a lower nucleosomal barrier and nucleosome translocation during transcription by Pol III-type mechanism.
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    ABSTRACT: In vitro transcription using the bacteriophage RNA poly-merase is an efficient way to synthesize RNA molecules in a large scale. There are several RNA polymerases and each enzyme needs a different DNA promoter sequence for initiation of transcription. 1-3 Among the RNA polymerases used to synthesize RNAs in vitro, bacteriophage T7 RNA polymerase (T7 RNAP) is the most widely used due to its convenience and ample synthesis of RNA using DNA tem-plate harboring conserved sequences of upstream promoter. T7 RNAP is a single subunit enzyme with a molecular weight of 98 kDa, which can catalyze RNA synthesis with-out any accessory proteins. 4-6 T7 RNAP binds to a variety of promoter sequences present in bacteriophage genome, all of which contain a 17-base-pair of consensus sequence as promoter. 6,7 In this study, forked DNA construct was design-ed, which contains the 5' and 3' overhangs comprised of thymine nucleotides and the 35-base-pair duplex harboring the T7 promoter sequence (Fig. 1(a)). The fidelity of the in vitro transcription process is usually sufficient for low scale synthesis of RNAs. However, non-templated 3'-extension incorporating extra nucleotides at the 3'-terminal of nascent RNA strands has been often re-cognized as a propensity of T7 RNAP, producing aberrant run-off transcripts in some cases. 8,9 Especially, these incorrect run-off transcripts are often generated in a relatively short RNA synthesis less than 20 bases. 9 Thus, oligoribonucleo-tide synthesis with defined sequences entails verification of exact length as well as 3'-terminal sequences. Since trans-location of the T7 RNAP along the DNA template is elemental for the nascent RNA synthesis, we hypothesized that T7 RNAP could be stalled at the end of DNA template without being released from the DNA template in the case of run-off products generation. This circumstance would make the T7 RNAP add a few more extra nucleotides to the transcript and generate aberrant run-off RNA products. T7 bacteriophage gene gp4A encodes another class of DNA translocating proteins, T7 DNA helicase, which is a 63-kDa replicative primase-helicase protein 10 that assembles into a ring-shaped hexamer in the presence of dTTP. 11-13 T7 helicase can form both hexamer and heptamer, but hexamer Figure 1. (a) Forked DNA template for both T7 RNAP and T7 helicase is shown, which contains T7 promoter sequence (indicated in a box) and two tails at the end of the template. In vitro transcription with the template generates aberrant RNA products as well as 14-mer RNA. +1 indicates initiation point of RNA synthesis. (b) Unwinding of the forked DNA substrate by T7 DNA helicase.
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