Single-molecule imaging of telomerase reverse transcriptase in human telomerase holoenzyme and minimal RNP complexes
- University of California, Berkeley, United States
Figures
Figure 1 with 1 supplement
Reconstitution, purification, and labeling of human TERT.
(A) Left: derivatized CoA Ppant prosthetic group transfer to acyl carrier protein (ACP) or MCP tag by ACP or SFP synthase. The MCP tag is a modified version of the ACP tag, containing two amino acid …
Figure 1—figure supplement 1
Methods of human telomerase reconstitution and purification.
(A) Telomerase reconstitutions in cells with full-length hTR or in RRL with hTRmin generate hTR-free TERT, TERT RNPs, and TERT-free hTR. These are differentially enriched by TERT-based vs …
Figure 2 with 1 supplement
Single-molecule detection of the TERT subunit content in DNA-bound complexes.
(A) Left: schematic for detection of TERT content by two-color co-localization. ACP-TERT was labeled with Cy5 (red) and MCP-TERT was labeled with Cy3 (green). PEG indicates polyethylene glycol. Cente…
Figure 2—figure supplement 1
Technical robustness of the two-color co-localization assay for TERT subunit content.
Quantification of two-color co-localization for DNA-bound 293T-F and 293T-O TERT complexes was consistent across different spot densities per field (A) and using cell extracts from independent …
Figure 3
Quantification of the TERT monomer vs multimer content in purified samples based on TERT-labeling efficiency.
(A) SDS-PAGE analysis of the kinetics of labeling F-purified 293T- or RRL-reconstituted MCP-TERT in reactions with CoA-Cy5 or CoA-Cy3 and SFP synthase. Lines within the panel indicate separate sets …
Figure 4
Distinct profiles of activity-dependent elution across populations of TERT complexes.
(A) Schematic of TERT complexes' interaction with bound DNA. In the presence of dTTP and dATP, complexes bound productively to primer end GGG-3′ would elongate the primer to GGGTTA-3′ accompanied by …
Figure 5
Direct DNA-binding affinity comparison for TERT complexes in bulk purifications.
(A) Schematic for detection of Cy5-labeled DNA binding to biotinylated TERT complexes. (B) SDS-PAGE analysis of MCP-TERT complexes labeled using CoA-Cy5. Cy5-labeled MCP-TERT proteolysis products …
Figure 6
Telomerase RNP assembly and activity without the TEN domain linker.
(A) Schematic representation and expression of N-terminally F-tagged human TERT proteins with the linker replaced by 20, 10, or 5 repeats of the sequence NAAIRS (TERT-20N through 5N) or linker …
Figure 7
PAL-mediated TERT dimerization.
(A) Two-color co-localization quantification for DNA-bound O-purified complexes of coexpressed ACP- and MCP-TERTs. Values are the average of triplicate experimental replicates. ***p < 0.001 using …
