Stepwise protein-mediated RNA folding directs assembly of telomerase ribonucleoprotein

Stone, Michael D.; Mihalusova, Mariana; O’Connor, Catherine; Prathapam, Ramadevi; Collins, Kathleen; Zhuang, Xiaowei

doi:10.1038/nature05600

Cited by 170 publications

(172 citation statements)

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“…3A) and allowed us to image single molecules. Although this anchoring scheme disrupted the loop at the distal end of stem II, previous studies have shown that extension, mutation, and disruption of the distal stem loop II do not inhibit enzyme assembly and function (16,26,27). Protein-free, stem-A-labeled and stem-B-labeled telomerase RNA gave rise to unexpected FRET values of 0.55 and 0.29, respectively (Fig.…”

Section: Resultsmentioning

confidence: 99%

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Functional importance of telomerase pseudoknot revealed by single-molecule analysis

Mihalusova¹,

Wu²,

Zhuang

2011

Proc. Natl. Acad. Sci. U.S.A.

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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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Section: Resultsmentioning

confidence: 99%

“…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)(15)(16).…”

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confidence: 99%

Functional importance of telomerase pseudoknot revealed by single-molecule analysis

Mihalusova¹,

Wu²,

Zhuang

2011

Proc. Natl. Acad. Sci. U.S.A.

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“…For a better understanding of how RT interacts with substrates, we designed a single-molecule assay to measure the enzyme orientation relative to its substrate by using fluorescence resonance energy transfer (FRET) 23,24 , a method well suited for probing dynamic interactions between proteins and nucleic acids [25][26][27] . Static FRET measurements have also been used previously to characterize the pre-translocation and post-translocation states of RT on a DNA duplex 28 .…”

Section: Single-molecule Assay For Enzyme-substrate Interactionsmentioning

confidence: 99%

Dynamic binding orientations direct activity of HIV reverse transcriptase

Abbondanzieri¹,

Bokinsky²,

Rausch

et al. 2008

Nature

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173

216

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The reverse transcriptase of human immunodeficiency virus (HIV) catalyses a series of reactions to convert the single-stranded RNA genome of HIV into double-stranded DNA for host-cell integration. This task requires the reverse transcriptase to discriminate a variety of nucleic-acid substrates such that active sites of the enzyme are correctly positioned to support one of three catalytic functions: RNA-directed DNA synthesis, DNA-directed DNA synthesis and DNA-directed RNA hydrolysis. However, the mechanism by which substrates regulate reverse transcriptase activities remains unclear. Here we report distinct orientational dynamics of reverse transcriptase observed on different substrates with a single-molecule assay. The enzyme adopted opposite binding orientations on duplexes containing DNA or RNA primers, directing its DNA synthesis or RNA hydrolysis activity, respectively. On duplexes containing the unique polypurine RNA primers for plus-strand DNA synthesis, the enzyme can rapidly switch between the two orientations. The switching kinetics were regulated by cognate nucleotides and non-nucleoside reverse transcriptase inhibitors, a major class of anti-HIV drugs. These results indicate that the activities of reverse transcriptase are determined by its binding orientation on substrates.Virtually all RNA-processing and DNA-processing enzymes show selectivity for backbone compositions or base sequences of their nucleic-acid substrates. This substrate selectivity is especially crucial for the HIV-1 reverse transcriptase (RT), which binds and discriminates between a variety of nucleic-acid duplexes for distinct catalytic functions 1,2 . RT is a heterodimer consisting of a p51 and a p66 subunit, the latter of which contains catalytically active DNA polymerase and RNase H domains 3,4 , catalysing a complex, multi-step reaction to convert the single-stranded RNA genome into double-stranded DNA 1,2 . First, RT uses the viral RNA genome as a template and a host-cell transfer RNA as a primer to synthesize a minus-strand DNA, producing an RNA-DNA hybrid [5][6][7] . This duplex becomes the substrate of the RNase H domain of RT, which cleaves the RNA strand at numerous points, leaving behind short RNA segments hybridized to the nascent DNA [8][9][10] . Among these RNAs, two specific purine-rich sequences, known as the polypurine tracts (PPTs), serve as unique primers to initiate the synthesis of plus-strand DNA 11-13 , thereby creating the double-stranded DNA viral genome. Specific cleavage by RNase H then removes the PPT primers and exposes the integration sequence to facilitate the insertion of the viral DNA into the host chromosome 14 . Inappropriate initiation of synthesis of the plus-strand DNA at other RNA segments prevents integration 2,15 . RT must therefore obey the following primer-selection rules: first, DNA primers readily engage the polymerase activity of RT; second, generic RNA primers are not efficiently extended by RT but readily engage the RNase H activity of RT when annealed with DNA; third, the PPT RNA ca...

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“…Fluorescence optical microscopy is a recently established method for the imaging of cellular structures, bacteria and viruses beyond the optical diffraction limit, down to a resolution of 6 nm. [1][2][3][4] This technique is based on the detection of light emitted by the fluorescing specimen when it is excited by light of a specific wavelength. Structured illumination, such as stimulated emission depletion and saturated structured illumination microscopy, which activate florescent light emission from a group of molecules simultaneously, are typically used.…”

Section: Introductionmentioning

confidence: 99%

Label-free super-resolution imaging of adenoviruses by submerged microsphere optical nanoscopy

et al. 2013

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Because of the small sizes of most viruses (typically 5-150 nm), standard optical microscopes, which have an optical diffraction limit of 200 nm, are not generally suitable for their direct observation. Electron microscopes usually require specimens to be placed under vacuum conditions, thus making them unsuitable for imaging live biological specimens in liquid environments. Indirect optical imaging of viruses has been made possible by the use of fluorescence optical microscopy that relies on the stimulated emission of light from the fluorescing specimens when they are excited with light of a specific wavelength, a process known as labeling or self-fluorescent emissions from certain organic materials. In this paper, we describe direct white-light optical imaging of 75-nm adenoviruses by submerged microsphere optical nanoscopy (SMON) without the use of fluorescent labeling or staining. The mechanism involved in the imaging is presented. Theoretical calculations of the imaging planes and the magnification factors have been verified by experimental results, with good agreement between theory and experiment.

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Stepwise protein-mediated RNA folding directs assembly of telomerase ribonucleoprotein

Cited by 170 publications

References 26 publications

Functional importance of telomerase pseudoknot revealed by single-molecule analysis

Functional importance of telomerase pseudoknot revealed by single-molecule analysis

Dynamic binding orientations direct activity of HIV reverse transcriptase

Label-free super-resolution imaging of adenoviruses by submerged microsphere optical nanoscopy

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