Nanopore tweezers show fractional-nucleotide translocation in sequence-dependent pausing by RNA polymerase

Abstract

RNA polymerases (RNAPs) carry out the first step in the central dogma of molecular biology by transcribing DNA into RNA. Despite their importance, much about how RNAPs work remains unclear, in part because the small (3.4 Angstrom) and fast (~40 ms/nt) steps during transcription were difficult to resolve. Here, we used high-resolution nanopore tweezers to observe the motion of single Escherichia coli RNAP molecules as it transcribes DNA ~1,000 times improved temporal resolution, resolving single-nucleotide and fractional-nucleotide steps of individual RNAPs at saturating nucleoside triphosphate concentrations. We analyzed RNAP during processive transcription elongation and sequence-dependent pausing at the yrbL elemental pause sequence. Each time RNAP encounters the yrbL elemental pause sequence, it rapidly interconverts between five translocational states, residing predominantly in a half-translocated state. The kinetics and force-dependence of this half-translocated state indicate it is a functional intermediate between pre- and post-translocated states. Using structural and kinetics data, we show that, in the half-translocated and post-translocated states, sequence-specific protein-DNA interaction occurs between RNAP and a guanine base at the downstream end of the transcription bubble (core recognition element). Kinetic data show that this interaction stabilizes the half-translocated and post-translocated states relative to the pre-translocated state. We develop a kinetic model for RNAP at the yrbL pause and discuss this in the context of key structural features.

Keywords: enzymology; nanopore tweezers; transcription; transcription elongation; transcription pausing.

Fig. 1.

Using nanopore tweezers to monitor translocation of RNAP on DNA in transcription elongation. (A) Experimental design. (A, Left) Backtracked, arrested TECs are assembled on synthetic nucleic-acid scaffolds containing the T7 A1 +27 arrest site and containing a single-stranded 3′ extension of the DNA template-strand to serve as a “handle” to enable capture of the DNA template-strand by the nanopore. (A, Right) Subpanel: Assisting force from voltage across the nanopore first draws the DNA template-strand (tDNA) into the nanopore until RNAP comes to rest on the rim of the nanopore and then triggers RNAP forward translocation, arrest escape, and transcription elongation, via the force on the DNA. During transcription elongation, translocation of RNAP relative to DNA controls the passage of template-strand DNA through the nanopore, and the nanopore reads the sequence of the passing DNA template-strand. (B) tDNA sequence used in this study. Dashed boxes indicate the section of tDNA passing through the pore in the data below. Representative ion current vs. time trace. Ion current amplitudes are determined by the DNA sequence passing through the nanopore. (C, Left) Zoom-in of the dashed box in B. Each state is assigned to a position in the sequence by alignment to the average pattern of ion-current states. (D) Representative position-vs.-time traces. Indicated are backward steps and the dwell time.

Fig. 2.

Nanopore-tweezers analysis of processive transcription elongation kinetics and determination of the “registration distance” between the MspA constriction and RNAP active-center nucleotide-addition site. (A) Transcription elongation kinetics on pause-free sequences at saturating [NTPs] ([ATP] = [CTP] = [GTP] = [UTP] = 1,000 µM; black; N = 57 molecules) and limiting [ATP] and saturating of other NTPs ([ATP] = 10 µM, [CTP] = [GTP] = [UTP] = 1,000 µM; red; N = 34 molecules). (A) T_dwell (B) P_back, and (C) T_total at each sequence position. The x-axis in each plot shows the RNA nucleotide to be incorporated in the active site using the inferred 17 nt registration distance. Gaps indicate sequence positions with low ion-current contrast where kinetic measurements are not made. Error bars are SEM. (D) Illustration of nanopore-engaged TEC in the posttranslocated state, with the 17 nt registration distance indicated.

Fig. 3.

Nanopore-tweezers analysis of processive transcription elongation: association of nanopore position states with TEC states. (A) Illustration of nanopore-engaged TECs in RNAP in Back, Pre, Post, and Hyper. (B) Kinetic models of RNAP transcription at saturating [NTPs] (Top) and limiting [ATP] with saturating [CTP], [GTP], and [UTP] (Bottom). At limiting [ATP], the transition from posttranslocated state to pretranslocated state is much slower at sites requiring ATP incorporation into the RNA (red shaded box), compared to template positions requiring CTP, GTP, or UTP incorporation, enabling identification of nanopore tweezers states with transcription states. (C) Example position-vs.-time traces (two each) of RNAP in which a single NTP has been reduced to 10 µM, with all others at saturating conditions, 1,000 µM. (Top two: limiting [ATP]; Middle two: limiting [UTP]; Bottom two limiting [GTP]).

Fig. 4.

Nanopore-tweezers analysis of processive transcription elongation: response of TECs to assisting force during processive transcription elongation. Probability of transitioning from Post to Pre (A), Post to Escape (B), and Post to Hyper (C) for three sequence positions (position 21, red; position 24, green; position 29, blue) as a function of the applied voltage. (D) Kinetic model of processive transcription elongation: from Post, RNAP can transition backward to Pre or forward to Hyper. Eventually, an NTP is incorporated into the RNA in Post, enabling Escape to the next transcription position. N given in SI Appendix, Table S4, typically N ~ 10 to 30 RNAP molecules per condition.

Fig. 5.

Nanopore-tweezers analysis of sequence-dependent elemental pausing (epTEC). Detection of a half-translocated state at the yrbL pause sequence. (A) TECs in Pre, Half, and Post at a consensus elemental pause sequence. (B) DNA position-vs.-time trace at the yrbL elemental pause sequence. During yrbL-pausing we observe rapid transitions between five different kinetic states: Back, Pre, Half, Post, Hyper. Each of the five states is indicated, with each data point assigned to a transcription state based on a five-state HMM. The pause begins with the first visit to the half-translocated state and ends with the last visit to Post. (C) Cumulative distribution function of the dwell-time distribution for position state 47, conditioned on whether the visit was the instance preceding the first visit to position 47.5 (green), last (blue), or any other (red). (D) Dwell time of first (green), last (light blue), and other (red) visits to position 47.5. (E) Same as C, but for position 48. N = 61 enzymes at saturating [NTP] and 180 mV applied voltage (SI Appendix, Table S2).

Fig. 6.

Nanopore-tweezers analysis of sequence-dependent pausing: kinetics, substrate concentration dependence, and force dependence of pausing at the yrbL consensus elemental pause sequence. (A) Kinetic model of yrbL pausing. (B) Fraction of the pause lifetime spent in each translocational state. (C) Pause lifetime vs. incoming GTP concentration ([GTP]) at various assisting forces (120 mV, blue; 150 mV, black; 180 mV, green; 220 mV, pink). (D) Probability that a given visit to Post results in escape vs. [GTP] at assisting forces ranging from ~24 to 40 pN. (SI Appendix, Fig. S22). (E) Dwell time vs. voltage for Pre (orange), Half (yellow), and Hyper (burgundy). Gray lines and 1 SEM shaded areas based on fits to Eqs. 1 and 2. (F) Probability of backward step vs. voltage for Pre (orange) and Half (yellow). Error bars are SEM. N given in SI Appendix, Table S2.

Fig. 7.

Kinetics of mutant D446A RNAP. (A) TEC in Post at the yrbL consensus elemental pause sequence. RNAP βD446 is positioned to interact with G_CRE, located in nontemplate DNA strand at the downstream edge of the transcription bubble in the posttranslocated state. (B) Cumulative distribution of pause lifetimes for RNAP (blue) and RNAP^βD446A (red). (C) Dwell-time distributions for RNAP (blue, N = 61 enzymes) and RNAP^βD446A (red, N = 58 enzymes). Dwell times and transition probabilities are indicated below. * indicates a z-score > 2 and **z > 4, where z is defined by the absolute value of the difference in means divided by the square root of the sum of squared errors. βD446A substitution destabilizes Half and Post, resulting in decreased dwell times in Half and Post, an increased probability of backward steps, and a decreased probability of escaping the pause site on visits to Post. Error bars are SEM. (D) Crystal structures of transcription complexes in Pre (Left), Half (Center), and Post with bound nucleotide substrate. The DNA template-strand, the DNA nontemplate-strand, and the RNA strand are in black, gray, and magenta, respectively, with the nucleotides corresponding to the templating nucleotide of Post and its complementary partner highlighted in green. RNAP active-center catalytic Mg²⁺ ion, pink sphere; RNAP bridge helix, orange ribbon; RNAP β pocket and βD446 yellow; RNAP β fork loop and βR422, cyan. RNAP residues are numbered as in E. coli RNAP. G_CRE (green) flips outward to interact with βD446 (yellow) in the transition from Pre to Half. The red asterisk indicates the transition from Pre to Half to Post, revealing the two successive 0.5 nt translocations of RNAP relative to the DNA template strand.