FIGURE 4.

ADP-ribosylation by HopU1 reduces ability of GRP7 to bind RNA. A, electrophoretic mobility shift assay of an RNA probe with GRP7-GST after treatment with HopU1-His or its catalytic inactive mutant HopU1_DD-His. Standard ADP-ribosylation reactions were performed with GRP7-GST or GRP7-GST_R49K in the presence of HopU1 or HopU1_DD, and a ³²P-labeled probe (ATGRP7 UTR WT) was added to each reaction mix. These were run on native polyacrylamide gels and exposed to x-ray films. B, similar assays done with differing amounts of GRP7-GST. The protein-bound and free RNA probes were quantified using a PhosphorImager scanner. The ratio of protein-bound and free forms of RNA was plotted against the concentration of GRP7 using a logarithmic scale. The x intercept allowed estimation of K_d for both HopU1 and HopU1_DD as indicated. The autoradiogram used to calculate the kDa is shown in supplemental Fig. S4.