Supplementary Figure 1 Telomerase RNA fragments used in single-molecule FRET experiments. A pseudoknot fragment (nts ) labeled at position U42

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1 Supplementary Figure 1 Telomerase RNA fragments used in single-molecule FRET experiments. A pseudoknot fragment (nts ) labeled at position U42 with Cy3 (green circle) was constructed by a two piece DNA-splinted RNA ligation at the indicated site. Based upon previously reported work, a U32G mutation was made to enhance P1 stem stability 1. A doubly-labeled pseudoknot fragment with an extended P1 stem harboring U42-Cy3 and U29- Cy5 labels was constructed by two sequential two piece DNA-splinted RNA ligation reactions as described in the methods. All telomerase reconstitution reactions included a separate CR4/CR5 (nts ) fragment generated by in vitro transcription as described in the methods. The secondary structure models are modified versions of a previous representation of human telomerase RNA 2.

2 Supplementary Figure 2 Quantification of telomerase enzyme reconstitution by RNA dot blot and direct DNA primer extension. a, (top) Scanned image of RNA dot blot quantification. (middle) Plot of internal standards from top panel quantified in ImageJ and fit by a linear regression. (bottom) Concentrations of telomerase extracted by standard curve. b, Direct DNA primer extension assays for all telomerase enzymes and DNA primers used in the study. Activity assays were performed as described in the detailed methods section unless otherwise noted. Concentrations of individual enzyme reconstitution reactions determined by RNA dot blot

3 analysis were used to calculate total enzyme activity, defined as the total counts per lane corrected for the recovery control (RC), relative to the wild type (WT) enzyme. (1) Unlabeled telomerase extension of 18GGG DNA primer. (2) Unlabeled telomerase extension of 5 - biotinylated 18GGG/T1 DNA primer. The retardation of products in this lane is likely due to the Cy5 label site being in close proximity to the 5 -biotin moiety. (3) Unlabeled telomerase extension of 18GGG/T7 DNA primer. (4) Unlabeled telomerase extension of 18GGG/T13 DNA primer. (5) U42-Cy3 labeled telomerase extension of 18GGG DNA primer. (6) Doubly-labeled U42-Cy3 and U29-Cy5 telomerase extension of 18GGG DNA primer. (7) U42-Cy3 labeled telomerase with UA47-48AU template mutation extension of 18GGG DNA primer demonstrates knockdown of repeat addition processivity. (Lanes 8-11) Single nucleotide addition and chain termination experiments which were accomplished by extension for 30 minutes to match smfret experiments described in Fig. 3 (main text). (8). Unlabeled telomerase extension of 17AGG primer in the presence of datp, dgtp [ 32 P], and ddttp yielding the product 19GGT. (9). Unlabeled telomerase extension of 17AGG primer in the presence of dgtp [ 32 P] and dttp yielding the product 20GTT. This experimental condition did not induce a clean 20GTT product and was therefore excluded from single-molecule FRET experiments. (10). Unlabeled telomerase extension of 17AGG primer in the presence of dgtp [ 32 P], dttp, ddatp yielding the product 21TTA. (11). Unlabeled telomerase extension of 5 -biotinylated 17AGG primer. For this experiment an initial incubation in the presence of dgtp [ 32 P] for 2 minutes was followed by the addition of dttp, datp, and ddgtp yielding the product 22TAG. (12) Unlabeled telomerase extension of 5 -biotinylated 18GGG in the presence of saturating triplet state quencher, TROLOX. (13) Unlabeled telomerase extension of 5 -biotinylated 18GGG in the absence of the triplet state quencher, TROLOX. c, After quantification of the bands using SAFA 11b, the processivities were calculated using a previously reported method 3. In short, the fraction of DNA left bound (FLB) at each repeat band was determined. A linear fit of ln(1-flb) versus repeat number was plotted and fit by linear regression using the expression ln(1- FLB)=(0.693/R 1/2 )*(repeat number). Dividing by the slope of each line provides R 1/2, the processivity value, for the respective enzyme. All the processivity values were quite similar except for a slight knockdown in processivity for the doubly labeled telomerase and the expected knockdown in the UA47-48AU mutant enzyme.

4 Supplementary Figure 3 Telomerase immobilization is DNA-primer dependent. a, FRETlabeled telomerase was added to the sample chamber in the absence of 5 biotinylated DNA primer, showing only a background level of complexes imaged by a TIRF microscope. b, Preincubation of FRET-labeled telomerase enzyme with 5 biotinylated DNA primer increases immobilization efficiency via the biotin interaction with the streptavidin coated surface.

5 Supplementary Figure 4 In situ telomerase activity alters the observed distribution of smfret values. a, The U42-Cy3 labeled telomerase was bound to 18GGG/T13-Cy5, producing a unimodal FRET distribution (left most panel). Addition of datp, dttp, and dgtp, initiates telomerase DNA primer extension activity. After incubation times of 5 minutes (b) and 60 minutes (c) the FRET distributions trend toward progressively lower values. Notably, the very low FRET value in panel (c) is not due to photo-bleaching or enzyme dissociation since the telomerase enzyme is labeled with the Cy3 donor dye. Therefore, this non-zero FRET value implies the Cy5 dye on the primer is moving away from the RNA template region. d, If the experiment is performed in the absence of dntps no change in the FRET distribution is observed. e, Telomerase enzymes reconstituted with a catalytically dead mutant TERT (D868A) show a similar initial FRET distribution to the wild type enzyme centered at FRET = f, As expected, a 60 minute incubation in the presence of dntps does not alter the FRET distribution for the D868A mutant, supporting the interpretation that a shift in the observed FRET distribution is due to telomerase catalytic activity on the slide. All histograms in the figure are constructed from at least 750 individual molecules.

6 Supplementary Figure 5 Single-round repeat addition processivity experiments. a, The translocation efficiency of our reconstituted telomerase is not quantitative as demonstrated by the ratio of the 22TAG (single repeat) vs. 25GGT (single round translocation). b, We therefore devised a method to specifically enrich for complexes that have successfully completed a single round of translocation for smfret analysis. U42-Cy3 labeled telomerase was pre-bound to the low affinity 5 biotinylated 21TTA/L2-Cy5 DNA primer. Addition of dgtp and ddttp initiates completion of the first telomere DNA repeat, translocation, and additional synthesis of the next repeat until chain termination by ddttp incorporation. Note that only those complexes that have successfully translocated and extended to the high affinity 25GGT state will remain bound to the biotinylated primer in the presence of 100-fold excess of non-biotinylated high affinity 18GGG chase primer. In contrast, complexes that stall in the low affinity 22TAG state will be effectively competed by the non-biotinylated 18GGG chase primer, and will therefore not be efficiently immobilized on the microscope slide for smfret analysis. c, This approach is validated by the observed ~2.5-fold increase in immobilization efficiency of complexes permitted to extend to the high-affinity 25GGT state compared to the low affinity 21TTA control. Bar plot shows average number of immobilized molecules per field of view in the microscope, and error bars represent the standard deviation across 20 independent fields of view.

7 Supplementary Figure 6 5 -DNA extrusion is coupled to translocation completion. a, The purity of synthetic primers 27PolyT-AGG, 31PolyT-TTA, and 32PolyT-TAG was tested by a 12% denaturing PAGE gel and ethidium bromide staining. The absence of impurities past the -1 band suggests the background non-specific products seen in Figure 6 Lanes 1 and 3 (main text), are due to a nuclease contaminant pulled down with telomerase. b, In Figure 5 (main text), we quantified the activity related bands (32 and 35 nt) and Exo related bands (17 and 20 nt) in Lanes 4-7, and compared the ratio of 35nt/32nt extension products to the ratio of 17nt/20nt ExoVII products. A correlation plot of these ratios as a function of the activity time course demonstrates a high level of correlation, strongly suggesting the 17 nt Exo VII cleavage product is derived from the 35 nt RAP product band. Error bars represent the standard deviation of the experiment performed in triplicate. c, To more directly verify the band at 17 nt was associated with the 35 nt translocated species, we used a primer that hybridizes to the full template repeat (32polyT-TAG). Extension of this primer in the presence of dgtp [ 32 P] and ddttp for 60 minutes dramatically enriches for the 35GGT RAP product band. When treated with Exo VII, we again observe two regions of protection centered about 17nt and 23nt (Lane 3). (Inset) Comparison of the ExoVII cleavage patterns from the 32 nt single repeat band (Lane 2, black line) and the 35 nt RAP product band (Lane 3, red line) de-convolutes the two sets of cleavage products that are superimposed in Figure 5c (main text).

8 Supplementary Table 1 DNA and RNA oligonucleotides Name 18GGG/T13 18GGG/T7 18GGG/T1 21TTA/T7 18GGG 17AGG U42 Labeled RNA U42 Labeled UA47-48-AU Mutant RNA U29 Labeled RNA DNA Splint for DNA Splint for PolyT-AGG 31PolyT-TTA 32PolyT-TAG htr PCR Primer Forward htr PCR Primer Reverse htr PCR Primer Forward htr PCR Primer Reverse htr PCR Primer Forward htr PCR Primer Reverse htr PCR Primer Forward htr PCR Primer Reverse Dot Blot Probe Primer Sequence (Label site*) 5 -Bio-TTAGGGTTAGGGTTAGGG-3 5 -Bio-TTAGGGTTAGGGTTAGGG-3 5 -Bio-TTAGGGTTAGGGTTAGGG-3 5 -Bio-TTAGGGTTAGGGTTAGGGTTA-3 5 -TTAGGGTTAGGGTTAGGG-3 5 -Bio-TTAGGGTTAGGGTTAGG-3 5 -GGGCCAUUUUUUGUCUAACCCUAACUGAGAA-3 5 -GGGCCAUUUUUUGUCAUACCCUAACUGAGAA-3 5 -GGGGUGAAC-3 5 -CAGCGCGCGGGGAGCAAAAGCACGGCGCCTACGCCCTTCT CAGTTAGGGTTAGACAAAAAATGGCCACCACCCCTCCCAGG-3 5 -CAGCGCGCGGGGAGCAAAAGCACGGCGCCTACGCCCTTCT CAGTTAGGGTTAGACAAAAAATGGCCCGTTCACCCC-3 5 -TTTTTTTTTTTTAGGGTTAGGGTTAGG-3 5 -TTTTTTTTTTTTAGGGTTAGGGTTAGGGTTA-3 5 -TTTTTTTTTTTTAGGGTTAGGGTTAGGGTTAG-3 5 -TAATACGACTCACTATAGGGCCATTTTTTGTCTAACCC-3 5 -AACGGGCCAGCAG-3 5 -TAATACGACTCACTATAGAGAAGGGCGTAGGC-3 5 -GGGGCGAACGGG-3 5 -TAATACGACTCACTATAGAGAAGGGCGTAGGC-3 5 -AACGGGCCAGCAG-3 5 -TAATACGACTCACTATAGAACCCCGCCTGG-3 5 -GACCCGCGGCTG-3 5 -CGGTGGAAGGCGGCAGGCCGAGGC-3 *Labeling sites in DNA or RNA oligonucleotides represent internal amino modifiers C6-dT (IDT) or 5-Amino-allyl-uridine (Dharmacon) respectively. These modified sites were then coupled to the appropriate FRET dye and purified by reverse phase HPLC.

9 Supplementary References 1. Chen, J. & Greider, C. W. Template boundary definition in mammalian telomerase. Genes Dev. 17, (2003). 2. Podlevsky, J. D., Bley, C. J., Omana, R. V, Qi, X. & Chen, J. J.-L. The telomerase database. Nucleic Acids Res. 36, D (2008). 3. Latrick, C. M. & Cech, T. R. POT1-TPP1 enhances telomerase processivity by slowing primer dissociation and aiding translocation. EMBO J. 29, (2010).