Figure 3.

The effect of OZM on translocation by Eco RNAP. (A) The nucleic acid scaffold employed in the translocation assay. The guanine analog 6-MI was initially positioned in the downstream DNA two nucleotides downstream of the active site. The 6-MI fluorescence was quenched by the neighboring base pairs in the initial TEC (state 1) and the pre-translocated TEC that formed following the nucleotide incorporation (state 2) but increases when the 6-MI relocates to the edge of the downstream DNA upon translocation (state 3). The Bridge Helix (BH) and the Lid loop (LL) are two structural elements of β’ subunit that flank the RNA:DNA hybrid in the multisubunit RNAPs. (B) The fluorescence intensities observed upon incorporation of the uridine or OZM or Ψ by TECs assembled with the wild-type Eco RNAP. Gel panels reveal the length of the RNA at each step. The incorporation of OZM resulted in a very small change in the mobility of the RNA but was verified by further extension of the TEC with the guanosine. (C) The experiments were performed as in (B), but the TECs were assembled using β’N458S Eco RNAP.