Dissociation of halted T7 RNA polymerase elongation complexes proceeds via a forward-translocation mechanism

Abstract

A recent model for the mechanism of intrinsic transcription termination involves dissociation of the RNA from forward-translocated (hypertranslocated) states of the complex [Yarnell WS, Roberts JW (1999) Science, 284:611-615]. The current study demonstrates that halted elongation complexes of T7 RNA polymerase in the absence of termination signals can also dissociate via a forward-translocation mechanism. Shortening of the downstream DNA or the introduction of a stretch of mismatched DNA immediately downstream of the halt site reduces a barrier to forward translocation and correspondingly reduces the lifetime of halted complexes. Conversely, introduction of a cross-link downstream of the halt site increases the same barrier and leads to an increase in complex lifetime. Introduction of a mismatch within the bubble reduces a driving force for forward translocation and correspondingly increases the lifetime of the complex, but only for mismatches at the upstream edge of the bubble, as predicted by the model. Mismatching only the two most upstream of the eight bases in the bubble provides a maximal increase in complex stability, suggesting that dissociation occurs primarily from early forward-translocated states. Finally, addition in trans of an oligonucleotide complementary to the nascent RNA just beyond the hybrid complements the loss of driving force derived from placement of a mismatch within the bubble, confirming the expected additivity of effects. Thus, forward translocation is likely a general mechanism for dissociation of elongation complexes, both in the presence and absence of intrinsic termination signals.

Fig. 1.

Forward translocation as a mechanism of elongation complex dissociation. Halted complexes distribute among various forward-translocated states, with complex dissociation being favored from those states with the smallest hybrids.

Fig. 2.

Contributions of downstream DNA to stability. (A) DNA constructs with varying lengths of downstream DNA. Sequences up to the halt site are identical for all constructs and allow halting at position +34 in the presence of GTP, ATP, and CTP. All constructs are fully duplex; the bubble in a halted complex is shown for clarity. In close-2 and close*-2, the end of the DNA is closed by an 18-atom polyethylene glycol spacer that covalently links the template and nontemplate strands, preventing full melting. (B) Denaturing gel electrophoresis of labeled RNA products at increasing time intervals. Reactions at 37°C contained 0.1 μM enzyme and 0.5 μM DNA (0.2/1.0 μM for the open*-2 and close*-2 constructs). (C) Stalled RNA products from B were quantified, and the data were plotted as a function of time. The slopes (velocities) divided by the concentration of enzyme were used as an estimate for the first-order rate constants (k_off) for dissociation, the rate limiting step in turnover. (D) The rate constants were then used to calculate the half-lives of the halted elongation complexes by using t_½ = ln(2)/k_off. (E) The nontemplate strand sequence of the control construct (others have the same sequence truncated appropriately). (E*) The nontemplate strand sequence of the constructs open*-2 and close*-2.

Fig. 3.

Reducing the downstream barrier to and the upstream driving force for forward translocation. (A) Constructs used. (B) Denaturing gel electrophoresis of reactions as described in Fig. 2. (C and D) Quantification of stability as described in Fig. 2.

Fig. 4.

Collapse of the upstream edge of the bubble helps to drive forward translocation. Placement of two- or three-base mismatches at different positions in the bubble demonstrates that stability derives from decreasing reannealing at the upstream edge only. (A) Shown are constructs used; control is fully matched. (B) Lifetimes of halted complexes on the indicated constructs. Reactions and analyses are the same as for Fig. 2.

Fig. 5.

Bumping does not require forward translocation. In order make bumping the dominant mechanism of dissociation, experiments were carried out as described in Fig. 2 but with concentrations of enzyme and DNA reversed (enzyme, 0.5 μM; DNA, 0.1 μM). (A) Denaturing gel electrophoresis of reactions. (B and C) Quantification of stability is the same as in Fig. 2. Note the much shorter lifetimes compared with previous figures.

Fig. 6.

Introduction of a new driving force complements the loss of upstream bubble collapse. Transcribing from template (up 2), which contains a mismatch at positions −8 and −7 in the bubble (see Fig. 4), an oligonucleotide complementary to bases −24 to −9 of the RNA transcript was added in ≈150-fold excess over DNA (to drive binding).