Extended Data Fig. 5. RNA primers initiate with ATP and are approximately 8 nt long.
a, Reactions with γ-32P-ATP (trace concentration) as the only labelled nucleotide, with increasing concentrations of unlabelled ATP as indicated. Label incorporation decreases at 100 and 200 µM rATP, because above Km, dilution of the radiolabelled rATP is no longer compensated by increased reaction velocity. b, Hydrolysis of the RNA primer with NaOH or RNase A shortens the dC-labelled C-strand. WT, wild-type CST–Polα-primase, T-End, 5’-end labeled telomerase reaction products as size markers. RNase A, reaction products from lane WT were boiled and then treated with RNase A. NaOH, reaction products from lane WT were treated with NaOH. The 8-nt reduction in dC-labeled product size upon NaOH treatment indicates that the C-strands initiate with pppAACCCUAA/dCdCdC…, where the RNA primer is underlined and the slash indicates the 3’-most site of hydrolysis. RNase A, which cleaves after pyrimidines, would then cleave as follows: pppAACCU/AAdCdCdC…. This would result in the RNase A products being two nt longer than the NaOH products, as observed. The lines connecting the bands on the gel indicate the reduction in product size upon NaOH or RNase A treatment. For gel source data, see Supplementary Fig. 1.