Characterization of T7 RNA Polymerase Transcription Complexes Assembled on Nucleic Acid Scaffolds

Abstract

We have used synthetic oligomers of DNA and RNA to assemble nucleic acid scaffolds that, when mixed with T7 RNA polymerase, allow the formation of functional transcription complexes. Manipulation of the scaffold structure allows the contribution of each element in the scaffold to transcription activity to be independently determined. The minimal scaffold that allows efficient extension after challenge with 200 mM NaCl consists of an 8-nt RNA primer hybridized to a DNA template (T strand) that extends 5-10 nt downstream. Constructs in which the RNA-DNA hybrid is less than or greater than 8 bp are less salt-resistant, and the hybrid cannot be extended beyond 12-13 bp. Although the presence of a complementary nontemplate strand downstream of the primer does not affect salt resistance, the presence of DNA upstream decreases resistance. The addition of a 4-nt unpaired "tail" to the 5 end of the primer increases salt resistance, as does the presence of an unpaired nontemplate strand in the region that contains the 8-bp hybrid (thereby generating an artificial transcription "bubble"). Scaffold complexes having these features remain active for over 1 week in the absence of salt and exhibit many of the properties of halted elongation complexes, including resistance to salt challenge, a similar trypsin cleavage pattern, and a similar pattern of RNA-RNA polymerase cross-linking.During the early stages of transcription, T7 RNAP 1 (like all known RNAPs) forms an unstable initiation complex (IC) that synthesizes and releases short abortive initiation products before clearing the promoter and forming a stable elongation complex (EC) (1-3). The transition is accompanied by release of upstream promoter contacts, changes in the size of the footprint of the polymerase on the DNA, increased resistance to challenge with agents such as salt and heparin (which disrupt and inactivate the IC), and changes in accessibility to cleavage by a variety of proteases (4 -7). Taken together, these changes suggest that significant alterations in the organization of the complex occur during the transition.A variety of lines of evidence demonstrate that the transition is complex and may involve multiple steps (6, 8 -13). Recent fluorescence probing and nuclease sensitivity experiments indicate that promoter clearance and collapse of the upstream edge of the transcription bubble occur when the RNA-DNA hybrid achieves a length of 8 -9 bp (13, 14) but that the length of the hybrid increases to 10 bp before the transcription bubble collapses to yield a hybrid length of 8 bp, as is observed during elongation (Ref. 13 and see below). The final phase (between 10 and 14 nt) appears to involve displacement of the 5Ј end of the RNA from the upstream end of the hybrid and its association with an RNA product-binding site (11, 15). Crystal structures have now been solved for free RNAP, for RNAP complexed with a specific inhibitor of transcription (T7 lysozyme), for a binary promoter-RNAP complex, and for an initiation complex that has transcribed the first thre...

Biochemical Analysis of Hypermutation by the Deoxycytidine Deaminase APOBEC3A

“…We hypothesized that Apo3G should be less able to deaminate dsDNA undergoing transcription due to its size. Apo3G is roughly twice the size of Apo3A and AID (46 kDa compared with 23 and 24 kDa, respectively), which could make access to the transcription bubble (ϳ9 nt) unlikely (66). As expected, Apo3G was not able to effectively catalyze deaminations during T7-mediated transcription (Fig.…”

New Insights into the Mechanism of Initial Transcription

“…Previous studies have exploited a proteolysis assay to follow the protein structural change associated with this transition (18,21,22). The proteolysis data presented in Fig.…”

Probing conformational changes in T7 RNA polymerase during initiation and termination by using engineered disulfide linkages

“…A nucleic acid scaffold that allows the formation of an EC by T7 RNAP (TS1͞NT1͞RNA12) has been described (21). Synthetic doublestranded templates that contain a T7 promoter were constructed by annealing complementary T and NT strands together at 70°C for 10 min followed by slow cooling to room temperature (22).…”