Figure 5.

Crosslinking of proteins specific to the TCE. (A) The constructs C2, C3, C7, and C8, which correspond to Prl-C2, Prl-C3, Prl-C7 and Prl-C8, respectively, but lack the 5′ UTR and coding regions, were used to synthesize ³²P-labeled RNA. An amount of each RNA corresponding to equal incorporated counts was incubated with Xenopus oocyte extract and subjected to UV-mediated crosslinking. The labeled proteins were separated by SDS/PAGE and visualized by autoradiography. The asterisk indicates the position of a 38-kDa protein that appears to be specifically crosslinked to RNAs containing the TCE. (B) Effect of substrate RNA titration on crosslinking. Various concentrations of ³²P-labeled C2 (○) and C3 (●) RNA were used for UV crosslinking as in A. The 38-kDa band was quantitated by densitometry (with local background subtraction) and plotted as a function of RNA concentration. (C) Binding of 38-kDa protein is competed by the TCE but not by the minimum localization element or globin. RNA corresponding to the globin coding region, localization element (LE), or TCE (C7) were synthesized and quantitated. A 50-fold excess of each was mixed with ³²P-labeled C7 RNA (0.25 nM final concentration), and UV crosslinking was performed as in A. The arrowhead indicates the position of the 38-kDa crosslink. Lane 1 is the crosslinking reaction containing no competitor RNA.