Conformation of Telomerase RNP Established through Footprinting and Single-Molecule FRET

Abstract

Telomerase, a ribonucleoprotein (RNP) expressed in all highly proliferating cells, is comprised of telomerase RNA, telomerase reverse transcriptase (TERT) and other protein cofactors. The complex compensates for the chromosomal shortening that arises with each round of DNA replication. Since mutations altering telomerase expression, assembly and regulation cause multiple human diseases, it is of utmost importance to understand telomerase at the structural level. However, while the structure of TERT in the absence of RNA has been recently published, the full RNP structure remains unknown.Using a combination of single-molecule FRET and ensemble footprinting, we are probing the functional structure of telomerase RNP. In particular, we have determined the active conformation adopted by two conserved regions of telomerase RNA - the stemloop IV and the pseudoknot - upon assembly with TERT and other protein cofactors.The pseudoknot, unformed in naked telomerase RNA, was folded in active RNPs. Proper pseudoknot folding was required for catalysis, as demonstrated by mutations that abolished telomerase activity when formation of either pseudoknot stem was disallowed. Conversely, compensatory mutations that reinstated basepairing in the pseudoknot region also restored telomerase activity.Binding of TERT, in addition to allowing pseudoknot formation, brought loop IV into the proximity of the pseudoknot without affecting its conformation. Mutations in the loop IV region that abolished TERT binding and telomerase activity resulted in an extended RNA conformation with a substantially larger loop IV-pseudoknot distance, as well as protection of the pseudoknot by RNP proteins. Interestingly, the telomerase holoenzyme protein p65 could compensate for these effects of loop IV mutations, restoring the loop IV-TERT interaction, the folded conformation of stemloop IV and telomerase activity.These results suggest that proper conformations of the pseudoknot and stemloop IV of telomerase RNA are critical for enzyme activity.