The interaction between the yeast telomerase RNA and the Est1 protein requires three structural elements

Abstract

In the budding yeast Saccharomyces cerevisiae, the telomerase enzyme is composed of a 1.3-kb TLC1 RNA that forms a complex with Est2 (the catalytic subunit) and two regulatory proteins, Est1 and Est3. Previous work has identified a conserved 5-nt bulge, present in a long helical arm of TLC1, which mediates binding of Est1 to TLC1. However, increased expression of Est1 can bypass the consequences of removal of this RNA bulge, indicating that there are additional binding site(s) for Est1 on TLC1. We report here that a conserved single-stranded internal loop immediately adjacent to the bulge is also required for the Est1-RNA interaction; furthermore, a TLC1 variant that lacks this internal loop but retains the bulge cannot be suppressed by Est1 overexpression, arguing that the internal loop may be a more critical element for Est1 binding. An additional structural feature consisting of a single-stranded region at the base of the helix containing the bulge and internal loop also contributes to recognition of TLC1 by Est1, potentially by providing flexibility to this helical arm. Association of Est1 with each of these TLC1 motifs was assessed using a highly sensitive biochemical assay that simultaneously monitors the relative levels of the Est1 and Est2 proteins in the telomerase complex. The identification of three elements of TLC1 that are required for Est1 association provides a detailed view of this particular protein-RNA interaction.

A second essential function of the Est1-binding arm of yeast telomerase RNA

“…1A; Seto et al 2002;Lubin et al 2012)-is highly conserved among budding yeasts (Dandjinou et al 2004;Lin et al 2004;Zappulla and Cech 2004). To further examine conservation in this region, we aligned all 36 publicly available distinct Saccharomyces TLC1 sequences (from seven different Saccharomyces species; Supplemental Fig.…”

“…The essential Est1 protein binds within the distal half of one arm, while the important Ku heterodimer and Sm 7 complexes bind to sites on the other arms (see Fig. 1A; Seto et al 1999Seto et al , 2002Peterson et al 2001;Stellwagen et al 2003;Lubin et al 2012). The Est3 protein has been reported to bind Est2 (Talley et al 2011) as well as Est1 (Tuzon et al 2011).…”

“…1A). TLC1 mutants lacking single-stranded RNA in this region have largely disrupted association with Est1 protein and exhibit short but stable telomeres (Lubin et al 2012). Together, these three structural elements provide the first essential function of the Est1 arm to be identified; namely, tethering Est1 protein to telomerase RNA.…”

“…1A; Seto et al 2003;Lubin et al 2012). The first of these regions comprises 5 nt predicted to form a bulge in the arm (Seto et al 2002).…”

“…The first of these regions comprises 5 nt predicted to form a bulge in the arm (Seto et al 2002). The second is within an internal loop closer to the tip of the Est1 arm; nucleotide sequence of the 5 ′ side of the loop is important and single-stranded RNA in the loop is critical for Est1 association with TLC1 (Lubin et al 2012). Two phylogenetically supported secondary structure models have been proposed for this internal loop; a single, large loop (Zappulla and Cech 2004) or two smaller loops separated by three A-U base pairs (Dandjinou et al 2004).…”