Telomerase is an essential cellular ribonucleoprotein (RNP) that solves the end replication problem and maintains chromosome stability by adding telomeric DNA to the termini of linear chromosomes [1][2][3] . Genetic mutations that abrogate normal assembly of telomerase RNP cause human disease 4 . It is thus of fundamental and medical importance to decipher cellular strategies for telomerase biogenesis, which will require new insights into how specific interactions occur in a precise order along the RNP assembly pathway. Here, we demonstrate a single-molecule approach to dissect the individual assembly steps of telomerase. Direct observation of complex formation in real time revealed two sequential steps of protein-induced RNA folding, establishing a hierarchical RNP assembly mechanism: interaction with the telomerase holoenzyme protein p65 induces structural rearrangement of telomerase RNA, which in turn directs binding of the telomerase reverse transcriptase (TERT) to form the functional ternary complex. This hierarchical assembly process is facilitated by an evolutionarily conserved structural motif within the RNA. These results identify the RNA folding pathway during telomerase biogenesis and define the mechanism of action for an essential telomerase holoenzyme protein.Telomerase RNP functions as a multi-subunit holoenzyme consisting of telomerase RNA, TERT, and additional protein cofactors. Catalytically active telomerase enzyme can be reconstituted from RNA and TERT in rabbit reticulocyte lysate (RRL) wherein general chaperone activities promote RNP assembly 5,6 . However, the endogenous process of telomerase biogenesis appears to require a more specific assembly pathway 7 . Supporting this view, cellular accumulation of telomerase RNP is promoted by a number of specific RNAbinding proteins, including dyskerin in vertebrates, Sm proteins in yeasts, and La-motif proteins in ciliates [8][9][10][11] . In this work, we exploited single-molecule fluorescence resonance energy transfer (FRET) 12-14 to explore the mechanism for telomerase RNP biogenesis, using the ciliate Tetrahymena thermophila as a model system.The Tetrahymena telomerase RNA is a 159 nucleotide transcript (Fig. 1a) that provides a template for telomere synthesis and functions in adapting the polymerase to its specialized taskCorrespondance and requests for materials should be addressed to X.Z., (E-mail: zhuang@chemistry.harvard.edu). Supplementary Information accompanies the paper on www.nature.com/nature. Competing interests statementThe authors declare that they have no competing financial interests. Fig. 1), we strategically placed a FRET donor (Cy3) and acceptor (Cy5) flanking the regions important for interaction with TERT and the holoenzyme protein p65, a La-motif protein that promotes telomerase RNP accumulation in vivo 11 (Fig. 1a). To facilitate real time observation of telomerase RNP assembly, RNA was surface immobilized through an extension of stem II that does not perturb telomerase activity in vitro or in vivo 19 . Standard ...

Telomerase ribonucleoprotein (RNP) employs an RNA subunit to template the addition of telomeric repeats onto chromosome ends. Previous studies have suggested that a region of the RNA downstream of the template may be important for telomerase activity and that the region could fold into a pseudoknot. Whether the pseudoknot motif is formed in the active telomerase RNP and what its functional role is have not yet been conclusively established. Using single-molecule FRET, we show that the isolated pseudoknot sequence stably folds into a pseudoknot. However, in the context of the full-length telomerase RNA, interference by other parts of the RNA prevents the formation of the pseudoknot. The protein subunits of the telomerase holoenzyme counteract RNA-induced misfolding and allow a significant fraction of the RNPs to form the pseudoknot structure. Only those RNP complexes containing a properly folded pseudoknot are catalytically active. These results not only demonstrate the functional importance of the pseudoknot but also reveal the critical role played by telomerase proteins in pseudoknot folding.T elomeres shorten with each round of DNA replication. Once telomeres reach a critical length, they become vulnerable to DNA damage response, which triggers cellular senescence and chromosome fusion (1). Telomerase protects telomeres from this replication-induced DNA erosion by addition of short G-rich repeats to the DNA ends (2). The telomerase enzyme is an attractive drug target in both cancer and regenerative medicine, because most highly proliferative cells, including stem and cancer cells, rely on its activity to maintain genomic integrity (3). Furthermore, multiple human diseases are known to be caused by mutations in telomerase subunits (4-6), underscoring the importance of understanding the enzyme's structure and function.The ciliate Tetrahymena thermophila has long been used as a model system to study telomerase activity. Tetrahymena telomerase is comprised of a 159-nucleotide RNA subunit, a 133-kDa telomerase reverse transcriptase (TERT), and multiple protein cofactors (7). The RNA has a conserved secondary structure (Fig. 1A) that contains a template region, which serves as template for the telomeric repeat synthesis, flanked upstream by the template boundary element (TBE) and downstream by a putative pseudoknot, as well as a highly structured region made of stems I and IV (8-10). TERT, the catalytic subunit, binds telomerase RNA in the TBE region, the template recognition element, and the loop of stem IV (10-13) (Fig. 1A) . Of the multiple Tetrahymena telomerase cofactors identified to date, only p65 binds the RNA directly in the stem I-proximal stem IV region (Fig. 1A) and promotes the hierarchical telomerase RNP assembly (14-16).Past studies have suggested that the putative pseudoknot region downstream of the template is a conserved and functionally important segment of telomerase RNA. Phylogenetic analyses of ciliate (9, 17), yeast (18-20), and vertebrate (21) RNAs suggest that this region may fold into a pse...

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