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. 2004 May;24(9):3720–3733. doi: 10.1128/MCB.24.9.3720-3733.2004

FIG. 4.

FIG. 4.

RID1 sequences that mediate repeat addition processivity are required for in vitro association with the hTR template-pseudoknot domain. (A) Schematic of the predicted hTR secondary structure (17, 20, 43). Numbers indicate nucleotide positions. The P1, P3, and P6.1 helices are indicated. (B) 32P-labeled wild-type (WT) hTR or hTR nucleotides 1 to 209 (top gel) were coimmunoprecipitated with Flag-tagged, 35S-labeled proteins (indicated by asterisks) corresponding to wild-type (WT) hTERT, RID2 (amino acids 300 to 617), or N- and C-terminally truncated RID1 variants (bottom gel). hTR association with RID1 and RID2 was expressed relative to hTR association with WT hTERT. αFlag, anti-Flag antibody. (C) The specificity of hTR1-209-RID1 interactions was determined by examining the interaction of a nonspecific Flag-tagged protein (NKKβ) or a nonspecific Drosophila RNA (DART-1) with hTR1-209 or Flag-tagged RID1, respectively. One microliter of a 1:500 dilution of an input RRL reaction mixture containing the appropriate 32P-labeled RNA was loadedin input lanes. PI, preimmune serum. (D) Results of competition experiments. 35S-labeled Flag-tagged RID1 and RID2 proteins were synthesized in RRL in the presence of equal amounts of 32P-labeled hTR1-209 and increasing amounts of unlabeled wild-type (WT) competitor hTR (1.48 × 10−21, 1.48 × 10−20, and 1.48 × 10−19 M). Ribonucleoprotein complexes were immunoprecipitated and visualized using the same method as other RNA binding experiments. One microliter of a 1:1,000 dilution of an input RRL reaction mixture containing 32P-labeled hTR1-209 was loaded in the input lane. αFlag, anti-Flag antibody; PI, preimmune serum; N.C., no competition (no competitor hTR). After normalization to the amount of protein in each lane, hTR1-209 association with RID1 or RID2 in the presence of competitor hTR was expressed relative to the no-competition (N.C.) control for each protein (percent association). Average interaction and standard deviation (Std. Dev.) values for different competitor hTR concentrations relative to N.C. controls are indicated at the bottom of each lane. At least three independent experiments for both RID1 and RID2 were performed.