Clusters of basic amino acids mediate L22 binding to EBER-1.

<p>(A) Binding of GFP-L22 to stem-loop III of EBER-1 (SL3) was tested in RNA EMSA experiments using increasing amounts of protein lysate (1, 5, and 20 µg) generated from 293T cells transfected with GFP-L22. 5 µg of control lysate expressing only GFP was used to assess nonspecific binding. Each 10 µl binding reaction contained 0.05 pmoles ³²P end-labeled SL3 RNA oligonucleotide. Reactions were electrophoresed on 8% native polyacrylamide gels and visualized by autoradiography. (B) L22 binds specifically to EBER-1. Binding specificity of L22 to SL3 was tested by antibody supershift and by competition with unlabeled oligonucleotides. 5 µg of GFP-L22 protein lysate was used in binding reactions. For antibody supershift experiments, 1 µl of anti-GFP or anti-polyhistidine (nonspecific control) antibody was added to binding reactions. In competition experiments, 10× and 100× unlabeled SL3 or mutated SL3 (mSL3) was added. (C) RNA binding capacity of GFP-L22 containing basic residue mutations or truncation of the amino-terminus (left panel) or with internal point mutations (right panel) was tested in RNA EMSA reactions, as described above. Amounts of each protein lysate used in binding reaction were determined by normalizing the level of expression of each mutated L22 construct to the level of GFP-L22 in 5 µg total protein lysate. Abbreviations used are: FP = free probe, NS = nonspecific, E = endogenous, GFP-L22 = all specific shifts generated with wild-type or mutated GFP-L22 proteins, SS = supershift.