Fig. 2.
Assembly of the chaperone complex between Imp3p, Imp4p and U3 MINI is RNA-dependent and may be ordered. (a) EFRET values are shown for three conditions: addition of Fl-Imp3p to a preincubated mixture of Rh-Imp4p and U3 MINI (filled bar); a mixture of Fl-Imp3p and Rh-Imp4p (hashed bar); and addition of Rh-Imp4p to a preincubated mixture of Fl-Imp3p and U3 MINI (open bar). (b) An emission spectrum of Rh-Imp4p added to a preincubated mixture of Fl-Imp3p and U3 MINI (solid line) illustrates the decrease in Fl emission (down arrow) with concomitant increase in Rh emission (up arrow) of a FRET signal. The emission spectrum of a preformed U3/Fl-Imp3p binary complex was the same in the presence (dashed line) and absence (data not shown) of unlabeled Imp4p, verifying that the signal is a result of FRET and not fluorescence quenching by Imp4p binding. (c) A schematic overview of the three steps of purification of the chaperone complex via metal affinity resin: (i) His6-Imp3p (grey), Imp4p (white), U3 snoRNA (black) or some combination thereof are loaded (L) onto a metal affinity resin; (ii) the column is washed; and (iii) the eluant is eluted (E) with addition of imidazole. (d) Four L and E fractions were analyzed on SDS-PAGE stained with ethidium bromide and silver nitrate to visualize the U3 snoRNA and the proteins, respectively.