Dataset Information

Yeast telomerase RNA: a flexible scaffold for protein subunits.

ABSTRACT: In the yeast Saccharomyces cerevisiae, distinct regions of the 1.2-kb telomerase RNA (TLC1) bind to the catalytic subunit Est2p and to accessory proteins. In particular, a bulged stem structure binds the essential regulatory subunit Est1p. We now show that the Est1p-binding domain of the RNA can be moved to three distant locations with retention of telomerase function in vivo. We present the Est1p relocation experiment in the context of a working model for the secondary structure of the entire TLC1 RNA, based on thermodynamic considerations and comparative analysis of sequences from four species. The model for TLC1 has three long quasihelical arms that bind the Ku, Est1p, and Sm proteins. These arms emanate from a central catalytic core that contains the template and Est2p-binding region. Deletion mutagenesis provides evidence that the Sm arm exists in vivo and can be shortened by 42 predicted base pairs with retention of function; therefore, precise positioning of Sm proteins, like Est1p, is not required within telomerase. In the best-studied ribonucleoprotein enzyme, the ribosome, the RNAs have specific three-dimensional structures that orient the functional elements. In the case of yeast telomerase, we propose that the RNA serves a very different function, providing a flexible tether for the protein subunits.

SUBMITTER: Zappulla DC

PROVIDER: S-EPMC454382 | biostudies-literature | 2004 Jul

REPOSITORIES: biostudies-literature

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Yeast telomerase RNA: a flexible scaffold for protein subunits.

Zappulla David C DC Cech Thomas R TR

Proceedings of the National Academy of Sciences of the United States of America 20040628 27

In the yeast Saccharomyces cerevisiae, distinct regions of the 1.2-kb telomerase RNA (TLC1) bind to the catalytic subunit Est2p and to accessory proteins. In particular, a bulged stem structure binds the essential regulatory subunit Est1p. We now show that the Est1p-binding domain of the RNA can be moved to three distant locations with retention of telomerase function in vivo. We present the Est1p relocation experiment in the context of a working model for the secondary structure of the entire T ...[more]

PMID: 15226497

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Project description:Saccharomyces cerevisiae telomerase RNA, TLC1, is an 1157 nt non-coding RNA that functions as both a template for DNA synthesis and a flexible scaffold for telomerase RNP holoenzyme protein subunits. The tractable budding yeast system has provided landmark discoveries about telomere biology in vivo, but yeast telomerase research has been hampered by the fact that the large TLC1 RNA subunit does not support robust telomerase activity in vitro. In contrast, 155-500 nt miniaturized TLC1 alleles comprising the catalytic core domain and lacking the RNA's long arms do reconstitute robust activity. We hypothesized that full-length TLC1 is prone to misfolding in vitro. To create a full-length yeast telomerase RNA, predicted to fold into its biologically relevant structure, we took an inverse RNA-folding approach, changing 59 nucleotides predicted to increase the energetic favorability of folding into the modeled native structure based on the p-num feature of Mfold software. The sequence changes lowered the predicted ∆G of this "determined-arm" allele, DA-TLC1, by 61 kcal/mol (-19%) compared to wild-type. We tested DA-TLC1 for reconstituted activity and found it to be ~5-fold more robust than wild-type TLC1, suggesting that the inverse-folding design indeed improved folding in vitro into a catalytically active conformation. We also tested if DA-TLC1 functions in vivo, discovering that it complements a tlc1∆ strain, allowing cells to avoid senescence and maintain telomeres of nearly wild-type length. However, all inverse-designed RNAs that we tested had reduced abundance in vivo. In particular, inverse-designing nearly all of the Ku arm caused a profound reduction in telomerase RNA abundance in the cell and very short telomeres. Overall, these results show that the inverse design of S. cerevisiae telomerase RNA increases activity in vitro, while reducing abundance in vivo. This study provides a biochemically and biologically tested approach to inverse-design RNAs using Mfold that could be useful for controlling RNA structure in basic research and biomedicine.

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Yeast telomerase RNA: a flexible scaffold for protein subunits.

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Yeast telomerase RNA: a flexible scaffold for protein subunits.

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