Effect of the G-quadruplex stability on the production of slipped and arrested transcripts.

<p>Denaturing gel electrophoresis of products of transcription reactions carried out for 90°C using Q5 template. Reaction mixtures contained 0.3 µM T7 polymerase and 1.5 µM DNA template in a buffer containing 40 mM Tris-HCl (pH 8.0), 8 mM MgCl₂, and 2 mM spermidine and various concentrations of (a) KCl or (b) LiCl. Lane 1 shows 10-nt size marker, lane 2 shows 35-nt RNA, and lanes 3 to 7 show transcription products in the presence of 0, 10, 30, 50, and 70 mM (a) KCl or (b) LiCl. Blue and red arrows indicate the slipped and arrested transcripts, respectively.</p

Effects on RNA polymerase elongation by structures in template DNA (a–d) and illustration of the template DNA (e, f).

<p>(a) An unstructured template, (b) a template with a slippage site, (c) a template with a pause site, and (d) a template with an arrest site. (e) The region denoted by the box marked with an X contains the sequence designed to form a random coil or non-canonical structure. (f) Sequence names and sequences of X regions. Sequences expected to form non-canonical structures are highlighted by italic and bold.</p

New Insights into Transcription Fidelity: Thermal Stability of Non-Canonical Structures in Template DNA Regulates Transcriptional Arrest, Pause, and Slippage

<div><p>The thermal stability and topology of non-canonical structures of G-quadruplexes and hairpins in template DNA were investigated, and the effect of non-canonical structures on transcription fidelity was evaluated quantitatively. We designed ten template DNAs: A linear sequence that does not have significant higher-order structure, three sequences that form hairpin structures, and six sequences that form G-quadruplex structures with different stabilities. Templates with non-canonical structures induced the production of an arrested, a slipped, and a full-length transcript, whereas the linear sequence produced only a full-length transcript. The efficiency of production for run-off transcripts (full-length and slipped transcripts) from templates that formed the non-canonical structures was lower than that from the linear. G-quadruplex structures were more effective inhibitors of full-length product formation than were hairpin structure even when the stability of the G-quadruplex in an aqueous solution was the same as that of the hairpin. We considered that intra-polymerase conditions may differentially affect the stability of non-canonical structures. The values of transcription efficiencies of run-off or arrest transcripts were correlated with stabilities of non-canonical structures in the intra-polymerase condition mimicked by 20 wt% polyethylene glycol (PEG). Transcriptional arrest was induced when the stability of the G-quadruplex structure (−ΔG^o₃₇) in the presence of 20 wt% PEG was more than 8.2 kcal mol⁻¹. Thus, values of stability in the presence of 20 wt% PEG are an important indicator of transcription perturbation. Our results further our understanding of the impact of template structure on the transcription process and may guide logical design of transcription-regulating drugs.</p></div

The stabilities of non-canonical structures designed to form in template DNA ^a

a<p>All experiments were carried out in a buffer containing 30 mM KCl, 40 mM Tris-HCl (pH 8.0), 8 mM MgCl₂, and 2 mM spermidine.</p>b<p>The sequences of template DNAs are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090580#pone-0090580-g001" target="_blank">Figure 1b</a> and Table S1 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090580#pone.0090580.s001" target="_blank">File S1</a>. The sequences designated by lower case letters contain only the non-canonical structure region (see Table S2 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090580#pone.0090580.s001" target="_blank">File S1</a>).</p>c<p>The melting temperature was determined at a strand concentration of 2 µM.</p>d<p>Arrest was defined as more than 4% production of arrested product RNA.</p>e<p>The −Δ<i>G</i>^o₃₇ value could not be determined because of very high stability.</p

Correlation between −Δ<i>G</i>^o₃₇ values obtained in 20 wt% PEG200 and transcription efficiency (TE).

<p>(a) TE of run-off transcripts and (b) TE of arrested transcript. Reaction mixtures contained 0.3 µM T7 polymerase and 1.5 µM DNA template in a buffer containing 40 mM Tris-HCl (pH 8.0), 8 mM MgCl₂, and 2 mM spermidine, and reactions were incubated for 90 min at 37°C.</p