Figure 6

Recombinant GST-PTB fusion is able to bind the IRES element: A) RNA-EMSA of wild type ³²P labeled IRES RNA with: lane I) ~50 μg of purified GST only, lane II) 0.3 μg of purified GST/PTB fusion protein and lane III) cold competition of RNA-protein complex formed in (II) by 20 fold molar excess of unlabeled RNA. The triangle indicates the position of the RNA-protein complex. B) UV cross-linking of GST-PTB to the IRES element. Lane I) GST plus wild type IRES element. Lane II) GST-PTB fusion protein plus wild type IRES element. Lane III) cold competition of (II) using 50 fold molar excess of unlabeled RNA. Lane IV) PPTDel1, lane V) PPTDel2 and lane VI) PPTDel3 IRES mutants. A RNA-protein complex of about 84 kDa was detected in all cases (see triangle), but significantly less in PPTDel2 and PPTDel3 deletion mutants. This complex corresponds to a fusion protein of PTB (57 kDa) and GST (27 kDa). Numbers on right site represent a protein molecular weight marker in kDa.