Structure of a Transcribing T7 RNA Polymerase Initiation Complex

Abstract

The structure of a T7 RNA polymerase (T7 RNAP) initiation complex captured transcribing a trinucleotide of RNA from a 17-base pair promoter DNA containing a 5-nucleotide single-strand template extension was determined at a resolution of 2.4 angstroms. Binding of the upstream duplex portion of the promoter occurs in the same manner as that in the open promoter complex, but the single-stranded template is repositioned to place the +4 base at the catalytic active site. Thus, synthesis of RNA in the initiation phase leads to accumulation or "scrunching" of the template in the enclosed active site pocket of T7 RNAP. Only three base pairs of heteroduplex are formed before the RNA peels off the template.

Functional Architecture of T7 RNA Polymerase Transcription Complexes

“…Effects of mutations in up-and downstream positively charged regions on EC stability. A: Transcripts (13mers) retained ("R") or freed ("F") from halted ECs formed with WT (lanes 1-6), K711C/K713E/K714E (lanes 7-12), K407E/K494E/K332E/K412C (lanes 13-18), or K407E/K412E (lanes 19-24) mutant T7RNAPs in the presence of 0 mM (lanes 1,2,7,8,13,14,19,20), 100 mM (lanes 3,4,9,10,15,16,21,22), or 800 mM (lanes 5,6,11,12,17,18,23,24) NaCl. B: Ratio of Free/Retained transcripts from the experiment in A plotted vs. enzyme and NaCl concentration (error bars give ranges from n=2).…”

T7 RNA polymerase transcription complex: What you see is not what you get

“…16). The proposed trajectory results in few clashes, is consistent with much of the biochemical and genetic data, and is not consistent with the RNA-exit pathway suggested by the structure of the T7 RNAP initiation complex (12). In an independent study, Shen and Kang (19) mapped T7 RNAP-RNA crosslinks from either the derivatized 3Ј end of the nascent RNA or from the Ϫ9 position of the nascent RNA in several stalled elongation complexes.…”

“…Thus, during slippage, the RNA-DNA hybrid is short, suggesting that the RNA-exit pathway of the complex engaged in the slippage synthesis is different from that in the productive complex (10). Interestingly, in bacterial RNAP a surface-exposed channel between the two domains of the second largest subunit exists (4) that is positioned analogously to the putative RNA-exit channel seen on the T7 RNAP transcribing complex (12). Consistent with this idea, in bacterial RNAP, mutations that alter the position of one of the second largest subunit domains or change residues that lie between the two domains result in dramatic changes in the efficiency of reiterative synthesis (24,25).…”

“…Thus, if the nucleic acid scaffold plays an essential role in transcription, one would expect that the sizes of the transcription bubble and RNA⅐DNA hybrid would be similar in transcription complexes formed by enzymes of both classes. It was surprising, therefore, when the structure of T7 RNAP complexed with a synthetic promoter and a trinucleotide RNA transcript revealed that only two RNA nucleotides closest to the catalytic center made Watson-crick interactions with the template strand of DNA (12). The 5Ј-proximal base of RNA appeared to peel away from the template, suggesting that in this system, the hybrid may be as short as 2-3 base pairs.…”

“…The RNA-DNA crosslinking experiment suggests a hybrid that is longer than observed in the structure of initiating T7 RNAP (12). To model the position of the hybrid in an elongating complex, Temiakov et al (15) mapped the site of a crosslink between a short-range crosslinker incorporated into the nascent RNA 9 base pairs upstream of the catalytic center and RNAP.…”