Fig. 2. Mapping of the RNA-binding region in hRUL138. (A) Deletion mapping. Crude lysates from bacteria expressing MBP-fused hRUL138 fragments truncated at the indicated positions (scheme above the gels; stippled bars outline hRUL138 parts not present) were tested for binding to a DIG-labeled HBV--containing RNA probe by Northwestern blotting. Bound RNA was visualized using anti-DIG antibody followed by chemiluminescent detection (left panel). The band at about 50 kDa is most likely a proteolytic hRUL138 fragment. Equal loading was confirmed by Coomassie blue staining of the SDS-PAGE gel (right panel) used to generate the blot. (B) Mutagenesis of the K-rich motif. Internal hRUL fragments amino acids 510 to 878 containing the authentic K-rich motif or the variant K4^- (amino acids 662 to 666=SGSTA) and amino acids 832 to 1176 were expressed in E. coli as fusions with the pET30 encoded linker, and processed as in A. For Northwestern analysis the blot was incubated with DIG-labeled HBV RNA (left panel); the bands below the 46 kDa marker position are probably degradation products. To control for loading, the same blot was reprobed with an antiserum recognizing the His-tag containing pET30 linker. (C) In-solution RNA binding. The same proteins as in B, except that a fragment comprising amino acids 1 to 205 was used as negative control, were incubated with ³²P-labeled HBV RNA, immobilized on Ni-NTA beads, and the radioactivity remaining on the beads was determined by scintillation counting. Results are shown as a percentage of the cpm measured for the authentic 510-878 fragment.