T7 RNA Polymerase Elongation Complex Research Articles

New Insights into the Mechanism of Initial Transcription

RNA polymerases undergo substantial structural and functional changes in transitioning from sequence-specific initial transcription to stable and relatively sequence-independent elongation. Initially, transcribing complexes are characteristically unstable, yielding short abortive products on the path to elongation. However, protein mutations have been isolated in RNA polymerases that dramatically reduce abortive instability. Understanding these mutations is essential to understanding the energetics of initial transcription and promoter clearance. We demonstrate here that the P266L point mutation in T7 RNA polymerase, which shows dramatically reduced abortive cycling, also transitions to elongation later, i.e. at longer lengths of RNA. These two properties of the mutant are not necessarily coupled, but rather we propose that they both derive from a weakening of the barrier to RNA-DNA hybrid-driven rotation of the promoter binding N-terminal platform, a motion necessary to achieve programmatically timed release of promoter contacts in the transition to elongation. Parallels in the multisubunit RNA polymerases are discussed.

Transcription Elongation Complex Stability

Transcription machinery from a variety of organisms shows striking mechanistic similarity. Both multi- and single subunit RNA polymerases have evolved an 8-10-base pair RNA-DNA hybrid as a part of a stably transcribing elongation complex. Through characterization of halted complexes that can readily carry out homopolymeric slippage synthesis, this study reveals that T7 RNA polymerase elongation complexes containing only a 4-base pair hybrid can nevertheless be more stable than those with the normal 8-base pair hybrid. We propose that a key feature of this stability is the topological threading of RNA through the complex and/or around the DNA template strand. The data are consistent with forward translocation as a mechanism to allow unthreading of the topological lock, as can occur during programmed termination of transcription.

Molecular dynamics studies of the energetics of translocation in model T7 RNA polymerase elongation complexes

Translocation in the single subunit T7 RNA polymerase elongation complex was studied by molecular dynamics simulations using the posttranslocated crystal structure with the fingers domain open, an intermediate stable in the absence of pyrophosphate, magnesium ions, and nucleotide substrate. Unconstrained and umbrella sampling simulations were performed to examine the energetics of translocations. The extent of translocation was quantified using reaction coordinates representing the average and individual displacements of the RNA-DNA hybrid base pairs with respect to a reference structure. In addition, an unconstrained simulation was also performed for the product complex with the fingers domain closed, but with the pyrophosphate and magnesium removed, in order to examine the local stability of the pretranslocated closed state after the pyrophosphate release. The average spatial movement of the entire hybrid was found to be energetically costly in the post- to pretranslocated direction in the open state, while the pretranslocated state was stable in the closed complex, supporting the notion that the conformational state dictates the global stability of translocation states. However, spatial fluctuations of the RNA 3'-end in the open conformation were extensive, with the typical range reaching 3-4 A. Our results suggest that thermal fluctuations play more important roles in the translocation of individual nucleotides than in the movement of large sections of nucleotide strands: RNA 3'-end can move into and out of the active site within a single conformational state, while a global movement of the hybrid may be thermodynamically unfavorable without the conformational change.

Imaging of T7 RNA polymerase elongation complexes by atomic force microscopy

Atomic force microscopy was used to visualize the complexes of bacteriophage T7 RNA polymerase (RNAP) with a DNA template during transcription. A 1414-bp fragment of linearized pGEMEX DNA was used as a template; the fragment contained the T7 promoter and terminator asymmetrically located at the template ends. Images of stable complexes of T7 RNAP and template terminal fragments were obtained for single molecules. Individual template DNA molecules bound with two or three T7 RNAP molecules, which corresponded to transcription initiation, elongation, and termination, and complexes containing RNA transcripts were imaged under conditions preventing nonspecific binding. The results suggest that T7 RNAP binds initially to the DNA template terminal fragment located near the promoter in order to exclude skipping the promoter site.

Observed Instability of T7 RNA Polymerase Elongation Complexes Can Be Dominated by Collision-induced “Bumping”

T7 RNA polymerase elongates RNA at a relatively high rate and can displace many tightly bound protein-DNA complexes. Despite these properties, measurements of the stability of stalled elongation complexes have shown lifetimes that are much shorter than those of the multisubunit RNA polymerases. In this work, we demonstrate that the apparent instability of stalled complexes actually arises from the action of trailing RNA polymerases (traveling in the same direction) displacing the stalled complex. Moreover, the instability caused by collision between two polymerases is position dependent. A second polymerase is blocked from promoter binding when a leading complex is stalled 12 bp or less from the promoter. The trailing complex can bind and make abortive transcripts when the leading complex is between 12 and 20 bp from the promoter, but it cannot displace the first complex since it is in a unstable initiation conformation. Only when the leading complex is stalled more than 20 bp away from the promoter can a second polymerase bind, initiate, and displace the leading complex.

Mapping the Conformation of the Nucleic Acid Framework of the T7 RNA Polymerase Elongation Complex in Solution Using Low-energy CD and Fluorescence Spectroscopy

The bacteriophage T7 elongation complex is an excellent model system in which to characterize the fundamental steps of transcription. We have formed functional elongation complexes, by mixing preassembled and RNA-primed DNA “bubble” constructs with T7 RNA polymerase and by initiating transcription at promoters, and have monitored the low-energy CD and fluorescence spectra of pairs of 2-aminopurine residues that have been inserted at defined sites within the DNA and RNA scaffold of the complex. In this way, we have been able to probe specific changes in the local conformations of the bases and base-pairs at these positions as the elongation complex goes through the various steps of the nucleotide addition cycle. The advantage of using pairs of 2-aminopurine residues, inserted at defined nucleic acid positions, as probes, is that the rest of the complex is spectrally “transparent” at wavelengths >300 nm. Thus, by combining CD and fluorescence measurements we obtain both structural and dynamic information that applies uniquely at each position within the functioning complex. In this way, we have mapped the details of steps central to transcription, including the formation and translocation of the transcription bubble, the formation and unwinding of the RNA–DNA hybrid, the passage of the nascent RNA through the exit channel of the polymerase, and the events of the template-controlled NTP selection process that controls transcriptional fidelity. This approach defines specific structural aspects of the elongation process under physiological conditions, and can be extended to examine other key aspects of transcriptional regulation, such as termination, editing, pausing, etc., that involve conformational rearrangements within the nucleic acid framework of the transcription complex.

Crystallization and preliminary crystallographic analysis of T7 RNA polymerase elongation complex.

Stable transcription-elongation complexes consisting of T7 RNA polymerase (molecular mass 99 kDa) in association with a nucleic acid scaffold consisting of an 8 bp RNA-DNA hybrid and 10 bp of downstream DNA were assembled and crystallized by the sitting-drop vapour-diffusion technique under near-physiological conditions. The crystals diffract beyond 2.6 A resolution and belong to space group P1, with unit-cell parameters a = 79.91, b = 84.97, c = 202 A, alpha = 90.36, beta = 92.97, gamma = 109.94 degrees. An unambiguous molecular-replacement solution was found using the C-terminal portion of the T7 RNA polymerase structure from the early initiation complex as a search model. Model building and structure refinement are now in progress.

Structure of a T7 RNA polymerase elongation complex at 2.9 A resolution.

The single-subunit bacteriophage T7 RNA polymerase carries out the transcription cycle in an identical manner to that of bacterial and eukaryotic multisubunit enzymes. Here we report the crystal structure of a T7 RNA polymerase elongation complex, which shows that incorporation of an 8-base-pair RNA-DNA hybrid into the active site of the enzyme induces a marked rearrangement of the amino-terminal domain. This rearrangement involves alternative folding of about 130 residues and a marked reorientation (about 130 degrees rotation) of a stable core subdomain, resulting in a structure that provides elements required for stable transcription elongation. A wide opening on the enzyme surface that is probably an RNA exit pathway is formed, and the RNA-DNA hybrid is completely buried in a newly formed, deep protein cavity. Binding of 10 base pairs of downstream DNA is stabilized mostly by long-distance electrostatic interactions. The structure implies plausible mechanisms for the various phases of the transcription cycle, and reveals important structural similarities with the multisubunit RNA polymerases.

T7 RNA polymerase elongation complex structure and movement

We have characterized T7RNAP elongation complexes (ECs) halted at different positions on a single template using a combination of digestion with exonuclease III, λ exonuclease, RNAse T1, and treatment with KMnO 4. Our results indicate that the transcription bubble is approximately nine bases long and that the RNA:DNA hybrid is 7-8 bp in size. An additional four to six bases of RNA immediately 5′ to the hybrid interact with the RNAP, probably with a site on the N-terminal domain. When ECs with transcripts of different length were probed in the presence or absence of the incoming NTP we found that the position of the EC on the template and the RNA shifted downstream upon NTP binding. NTP binding also restricted the lateral mobility of the complex on the template. Our results indicate that, in the absence of bound NTP, the RNAP is relatively free to slide on the template around a position that usually lies one to two bases upstream of the position from which NTP binding and bond formation occur. NTP binding stabilizes the RNAP in the post-translocated position and keeps it from sliding upstream, either due directly to RNAP:NTP:template interactions, or to an isomerization which causes the fingers subdomain of the RNAP to clamp down on the downstream end of the template strand.

Characterization of halted T7 RNA polymerase elongation complexes reveals multiple factors that contribute to stability

We have constructed a series of plasmid templates that allow T7 RNA polymerase (RNAP) to be halted at defined intervals downstream from its promoter in a preserved sequence context. While transcription complexes halted at +3 to +6 are highly unstable, complexes halted at +10 to +14 dissociate very slowly and gradually lose their capacity to extend transcripts. Complexes halted at +18 and beyond dissociate more readily, but the stability of the these complexes is enhanced significantly in the presence of the next incoming nucleotide. Unexpectedly, the stability of complexes halted at +14 and beyond was found to be lower on supercoiled templates than on linear templates. To explore this further, we used synthetic DNA templates in which the nature of the non-template (NT) strand was varied. Whereas initiation complexes are less stable in the presence of a complementary NT strand, elongation complexes are more stable in the presence of a complementary NT strand, and the presence of a non-complementary NT strand (a mismatched bubble) results in even greater stability. The results suggest that the NT strand plays an important role in displacing the nascent RNA, allowing its interaction with an RNA product binding site in the RNAP. The NT strand may also contribute to stabilization by interacting directly with the enzyme. A mutant RNAP that has a deletion in the flexible “thumb” domain responds to changes in template topology in a manner that is similar to that of the wild-type (WT) enzyme, but halted complexes formed by the mutant enzyme are particularly dependent upon the presence of the NT strand for stability. In contrast, an N-terminal RNAP mutant that has a decreased capacity to bind single-stranded RNA forms halted complexes with much lower levels of stability than the WT enzyme, and these complexes are not stabilized by the presence of the NT strand. The distinct responses of the mutant RNAPs to changes in template structure indicate that the N-terminal and thumb domains have quite different functions in stabilizing the transcription complex.

Transcription bypass or blockage at single-strand breaks on the DNA template strand: effect of different 3' and 5' flanking groups on the T7 RNA polymerase elongation complex.

We have studied the effects of single-strand breaks present on the template strand during T7 RNA polymerase transcription elongation. A synthetic DNA template with a T7 promoter was designed to contain a one-nucleotide gap at a defined location on the template strand. This gap, surprisingly, was efficiently bypassed by T7 RNA polymerase during transcription elongation, and the full-length transcript (FLT37) generated from the bypass event was shortened by one nucleotide compared to the full-length transcript (FLT38) generated from an intact, unbroken template strand. FLT37 did not contain any nucleotide insertions opposite to the gap, so that the RNA sequence downstream from the gap, although accurately transcribed, contained a single base deletion compared to FLT38. This, to our knowledge, is the first demonstration that the continuity of the DNA template strand is not a necessary requirement for DNA-dependent RNA polymerase transcription elongation. DNA templates with different 3' and 5' termini at the single-strand break site were also investigated in this study. One of these templates, 1/3P-4P, which contained 3'- and 5'-phosphoryl termini at the break site, efficiently blocked T7 RNA polymerase. A single phosphoryl group present on either the 3' or the 5' terminus of the break site did not efficiently block RNA polymerase progression, suggesting that the blockage observed with template 1/3P-4P is due to the repulsion between the two phosphoryl termini in the vicinity of the polymerase active site.