Structure in nascent RNA leads to termination of slippage transcription by T7 RNA polymerase

Abstract

T7 RNA polymerase presents a very simple model system for the study of fundamental aspects of transcription. Some time ago it was observed that in the presence of only GTP as a substrate, on a template encoding the initial sequence GGGA., T7 RNA polymerase will synthesize a 'ladder' of poly-G RNA products. At each step, the ratio of elongation to product release is consistently approximately 0.75 until the RNA reaches a length of approximately 13-14 nt, at which point this ratio drops precipitously. One model to explain this drop in complex stability suggests that the nascent RNA may be structurally hindered by the protein; the RNA may be exiting via a pathway not taken by normally synthesized RNA and therefore becomes sterically destabilized. The fact that the length of RNA at which this occurs is close to the length at which the transition to a stably elongating complex occurs might have led to other mechanistic proposals. Here we show instead that elongation falls off due to the cooperative formation of structure in the nascent RNA, most likely an intramolecular G-quartet structure. Replacement of GTP by 7-deaza-GTP completely abolishes this transition and G-ladder synthesis continues with a constant efficiency of elongation beyond the limit of detection. The polymerase-DNA complex creates no barrier to the growth of the nascent (slippage) RNA, rather termination is similar to that which occurs in rho-independent termination.

Figure 1

Minimal mechanism for slippage transcription. The efficiency of elongation at each step is the ratio of the elongation velocity to the sum of the velocities of the dissociation steps.

Figure 2

Comparison of the effects of 7-deaza-GTP on the synthesis of ‘G–-adder’ slippage products. (A) RNA products in a 10 min reaction at 37°C, containing 0.2 µM promoter DNA, 0.2 µM T7 RNA polymerase and 400 µM GTP (left) or 7-deaza-GTP (right), in a buffer of 20 mM HEPES pH 7.8, 25 mM potassium glutamate, 0.025% Tween-20, 2.5 mM Tris, 15 mM Mg(OAc)₂, 0.25 mM EDTA. Both reactions contained trace amounts (<0.06 µM) of [α-³²P]GTP for detection. The part of the gel showing the ‘regular’ G-ladder has been overexposed to make the difference between the two more apparent. (B) Structures of a G-quartet and of guanine and its 7-deaza analog. Note that replacement of guanine in the quartet structure by 7-deaza-guanine destroys the stability of the quartet. (C) Plots of percent fall-off as a function of the transcript length.

Figure 3

Effect of different fractional concentrations of 7-deaza-GTP on the termination of G-ladder synthesis. Conditions were as in Figure 2, except that varying amounts of 7-deaza-GTP replace GTP. In each case, the total concentration of nucleoside triphosphate (GTP plus 7-deaza-GTP) was 400 µM. Trace amounts (<0.06 µM) of [α-³²P]GTP were present in all lanes for detection.

Figure 4

(A) Time course of slippage synthesis with GTP as substrate. Conditions were as in Figure 2, except that in order to detect RNA at low turnover, concentrations of polymerase and promoter DNA were 5.0 and 3.0 µM, respectively. (B) Product concentrations at limited turnover. Data correspond to the reaction quenched at 10 s.