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. 2018 Feb 12;37(6):e97953. doi: 10.15252/embj.201797953

Figure EV3. K M measurement for nucleotide incorporations in the presence or absence of the pause signal.

Figure EV3

  1. Sequences of the DNA/RNA hybrid substrates, position of the pause signal, and pause site (red). In vitro‐reconstituted TF telomerase was analyzed with specific DNA/RNA hybrid substrates to determine the K M for each nucleotide incorporation. Numbers below the DNA/RNA duplexes indicate positions corresponding to the telomerase template and the order of nucleotides incorporated. Mutations to disrupt the pause signal denoted (orange). The DNA products were labeled by incorporating a 32P‐dATP (0.165 μM, asterisk) prior to the K M measurement.
  2. Representative gels for K M measurements. An 32P end‐labeled DNA recovery control (r.c.) was added before product purification and precipitation. The nucleotide incorporation K M values measured are indicated (black arrow).
  3. Plots derived from the normalized intensity of the denoted (black arrow) nucleotide incorporation product over the total intensity of products for the specified nucleotide concentration. The Michaelis–Menten equation, Y = V max*X/(K M+X), was used to fit the nonlinear curve to determine the K M. Two or three replicates were performed for each measurement.