FIGURE 3.

Influence of cofactors on formation of U2 stem IIa and IIc. (A–F) Histograms of E_FRET states observed for the WT RNA in the presence or absence of Mg²⁺ and/or Cus2p. Increasing concentrations of Mg²⁺ diminish the abundance of the high E_FRET values near 1.0 and increase the abundance of mid-E_FRET values. Addition of Cus2p causes the same effect and further reduces the number of high E_FRET values observed at 1 mM Mg²⁺. These data are consistent with Mg²⁺ and Cus2p suppressing stem IIc formation and increasing the abundance of stem IIa. (G–L) TODPs for data shown in A–F. Both Mg²⁺ and Cus2p induce patterns in the TODPs also observed when stem IIa was stabilized by mutation. Transitions to E_FRET states of ∼0.3 were suppressed in the presence of Cus2p. In G–L, data are colored as described in Figure 2. Each histogram and TODP represents data collected from N = 100–175 molecules.