Structure of Tetrahymena telomerase-bound CST with polymerase α-primase.

Telomeres are the physical ends of linear chromosomes. They are composed of short repeating sequences (such as TTGGGG in the G-strand for Tetrahymena thermophila) of double-stranded DNA with a single-strand 3' overhang of the G-strand and, in humans, the six shelterin proteins: TPP1, POT1, TRF1, TRF2, RAP1 and TIN21,2. TPP1 and POT1 associate with the 3' overhang, with POT1 binding the G-strand3 and TPP1 (in complex with TIN24) recruiting telomerase via interaction with telomerase reverse transcriptase5 (TERT). The telomere DNA ends are replicated and maintained by telomerase6, for the G-strand, and subsequently DNA polymerase α-primase7,8 (PolαPrim), for the C-strand9. PolαPrim activity is stimulated by the heterotrimeric complex CTC1-STN1-TEN110-12 (CST), but the structural basis of the recruitment of PolαPrim and CST to telomere ends remains unknown. Here we report cryo-electron microscopy (cryo-EM) structures of Tetrahymena CST in the context of the telomerase holoenzyme, in both the absence and the presence of PolαPrim, and of PolαPrim alone. Tetrahymena Ctc1 binds telomerase subunit p50, a TPP1 orthologue, on a flexible Ctc1 binding motif revealed by cryo-EM and NMR spectroscopy. The PolαPrim polymerase subunit POLA1 binds Ctc1 and Stn1, and its interface with Ctc1 forms an entry port for G-strand DNA to the POLA1 active site. We thus provide a snapshot of four key components that are required for telomeric DNA synthesis in a single active complex-telomerase-core ribonucleoprotein, p50, CST and PolαPrim-that provides insights into the recruitment of CST and PolαPrim and the handoff between G-strand and C-strand synthesis.

Tracking Mercury in Individual Tetrahymena Using a Capillary Single-Cell Inductively Coupled Plasma Mass Spectrometry Online System.

As a kind of unicellular eukaryotic protozoa, Tetrahymena is located at the bottom of the aquatic food webs and plays an essential role in the bioaccumulation of mercury (Hg). To track Hg in individual Tetrahymena, a capillary single-cell inductively coupled plasma mass spectrometry (ICPMS) online system was developed. The experimental and instrumental conditions were optimized to ensure the signal detected was the Hg uptake in individual Tetrahymena. Moreover, a quantitative method was established and validated by detecting Hg2+ standard solutions. The limit of quantity was calculated to be approximately 3.8 × 10-15 g Hg/cell, and the detection limit for Hg2+ exposure of Tetrahymena was 0.05 µg/L. By using the proposed method, we found the peak became wider with increasing of exposure concentrations, indicating the accumulated Hg by different Tetrahymena varied greatly, and the difference was more significant at higher exposure concentration. This novel method has the advantages of high sensitivity and real-time detection in individual Tetrahymena, and it could be widely used for further tracking the accumulation of mercury and other metals at the single cell level.

Tubulin lattice in cilia is in a stressed form regulated by microtubule inner proteins.

Cilia, the hair-like protrusions that beat at high frequencies to propel a cell or move fluid around are composed of radially bundled doublet microtubules. In this study, we present a near-atomic resolution map of the Tetrahymena doublet microtubule by cryoelectron microscopy. The map demonstrates that the network of microtubule inner proteins weaves into the tubulin lattice and forms an inner sheath. From mass spectrometry data and de novo modeling, we identified Rib43a proteins as the filamentous microtubule inner proteins in the protofilament ribbon region. The Rib43a-tubulin interaction leads to an elongated tubulin dimer distance every 2 dimers. In addition, the tubulin lattice structure with missing microtubule inner proteins (MIPs) by sarkosyl treatment shows significant longitudinal compaction and lateral angle change between protofilaments. These results are evidence that the MIPs directly affect and stabilize the tubulin lattice. It suggests that the doublet microtubule is an intrinsically stressed filament and that this stress could be manipulated in the regulation of ciliary waveforms.

Tetrahymena dynamin-related protein 6 self-assembles independent of membrane association.

Self-assembly on target membranes is one of the important properties of all dynamin family proteins. Drp6, a dynaminrelated protein in Tetrahymena, controls nuclear remodelling and undergoes cycles of assembly/disassembly on the nuclear envelope. To elucidate the mechanism of Drp6 function, we have characterized its biochemical and biophysical properties using size exclusion chromatography, chemical cross-linking and electron microscopy. The results demonstrate that Drp6 readily forms high-molecular-weight self-assembled structures as determined by size exclusion chromatography and chemical cross-linking. Negative stain electron microscopy revealed that Drp6 assembles into rings and spirals at physiological ionic strength. We have also shown that the recombinant Drp6 expressed in bacteria is catalytically active and its GTPase activity is not enhanced by low salt. These results suggest that, in contrast to dynamins but similar to MxA, Drp6 self-assembles in the absence of membrane templates, and its GTPase activity is not affected by ionic strength of the buffer. We discuss the self-assembly structure of Drp6 and explain the basis for lack of membrane-stimulated GTPase activity.

Tetrahymena Cilia Cap is Built in a Multi-step Process: A Study by Atomic Force Microscopy.

Cilia are complex and dynamic organelles that have motility and sensory functions. Defects in cilia biogenesis and function are at the origin of human ciliopathies. In motile cilia, a basal body organizes the axoneme composed of nine microtubule doublets surrounding a central pair of singlet microtubules. The distal ends of axonemal microtubules are attached to the membrane by microtubule-capping structures. Little is known about the early steps of cilium assembly. Although cilia grow and resorb from their distal tips, it remains poorly understood where and when the components of the caps are first assembled. By using Atomic Force Microscopy in tapping mode, with resolution at the nanometer range and with minimum sample manipulation, we show that Tetrahymena cilia assembly requires transient assembly of structures, composed of three components that are placed asymmetrically on an early elongating axoneme. In small uncapped axonemes the microtubule central pair was never observed. Additionally, we show that cilia cap assembly is a multi-step process in which structures of different sizes and shapes are put together in close proximity before the axoneme appears capped. We propose that the cap modifies the axoneme microtubule rate of polymerization and present a model for Tetrahymena cilia cap assembly.

The DEAD-Box Protein CYT-19 Uses Arginine Residues in Its C-Tail To Tether RNA Substrates.

DEAD-box proteins are nonprocessive RNA helicases that play diverse roles in cellular processes. The Neurospora crassa DEAD-box protein CYT-19 promotes mitochondrial group I intron splicing and functions as a general RNA chaperone. CYT-19 includes a disordered, arginine-rich "C-tail" that binds RNA, positioning the helicase core to capture and unwind nearby RNA helices. Here we probed the C-tail further by varying the number and positions of arginines within it. We found that removing sets of as few as four of the 11 arginines reduced RNA unwinding activity (kcat/KM) to a degree equivalent to that seen upon removal of the C-tail, suggesting that a minimum or "threshold" number of arginines is required. In addition, a mutant with 16 arginines displayed RNA unwinding activity greater than that of wild-type CYT-19. The C-tail modifications impacted unwinding only of RNA helices within constructs that included an adjacent helix or structured RNA element that would allow C-tail binding, indicating that the helicase core remained active in the mutants. In addition, changes in RNA unwinding efficiency of the mutants were mirrored by changes in functional RNA affinity, as determined from the RNA concentration dependence of ATPase activity, suggesting that the C-tail functions primarily to increase RNA affinity. Interestingly, the salt concentration dependence of RNA unwinding activity is unaffected by C-tail composition, suggesting that the C-tail uses primarily hydrogen bonding, not electrostatic interactions, to bind double-stranded RNA. Our results provide insights into how an unstructured C-tail contributes to DEAD-box protein activity and suggest parallels with other families of RNA- and DNA-binding proteins.

Posttranslational Modifications of Tubulin and Cilia.

Tubulin undergoes several highly conserved posttranslational modifications (PTMs) including acetylation, detyrosination, glutamylation, and glycylation. These PTMs accumulate on a subset of microtubules that are long-lived, including those in the basal bodies and axonemes. Tubulin PTMs are distributed nonuniformly. In the outer doublet microtubules of the axoneme, the B-tubules are highly enriched in the detyrosinated, polyglutamylated, and polyglycylated tubulin, whereas the A-tubules contain mostly unmodified tubulin. The nonuniform patterns of tubulin PTMs may functionalize microtubules in a position-dependent manner. Recent studies indicate that tubulin PTMs contribute to the assembly, disassembly, maintenance, and motility of cilia. In particular, tubulin glutamylation has emerged as a key PTM that affects ciliary motility through regulation of axonemal dynein arms and controls the stability and length of the axoneme.

Lectins and Tetrahymena - A review.

The unicellular ciliate Tetrahymena is a complete organism, one of the most highly developed protozoans, which has specialized organelles performing each of the functions characteristic to the cells of higher ranked animals. It is also able to produce, store, and secrete hormones of higher ranked animals and also react to them. It produces lectins that can bind them and has functions, which are influenced by exogenous lectins. The review lists the observations on the relationship between lectins and Tetrahymena and try to construe them on the basis of the data, which are at our disposal. Considering the data, lectins can be used by Tetrahymena as materials for influencing conjugation, for stimulating hormone receptors, and by this, mimic the hormonal functions. Lectins can influence phagocytosis and movement of the cells as well as the cell division. As Tetrahymena can recognize both related and hostile cells by the help of lectins and surface sugars, it could be surmised a complex predator-prey system. This could determine the survival of the population as well as the nourishment conditions. When Tetrahymena is pathogenic, it can use lectins as virulence factors.

Calculation of the HOMO localization of Tetrahymena and Oxytricha telomeric quadruplex DNA.

Several guanine-rich sequences exist in many important regions, such as telomeres, and these sequences can form quadruplex DNA structures. It was previously reported that 3'-guanines are mainly oxidized in the Tetrahymena and Oxytricha telomeric quadruplex DNA, d(TGGGGT)4, and 5'-guanines are mainly oxidized in the human telomeric quadruplex DNA, d(TAGGGT)4T. We speculated that the differences in site reactivity between d(TGGGGT)4 and d(TAGGGT)4T are induced by the localization of the HOMO. The HOMOs of the possible quadruplex structures were thus determined and the results showed that the HOMOs of d(TGGGGT)4 +3K(+) and d(TAGGGT)4T +2K(+) localized at the 5'-guanine, and that the HOMO shifted from the 5'-guanine to the 3'-guanine by the addition of a 5'-capping cation. Furthermore, we determined the influence of the cation and demonstrated that localization of the HOMO at the G-quartet plane located immediately adjacent to the cation is disfavored. The calculated HOMO localization of d(TGGGGT)4 +4K(+) and d(TAGGGT)4T +2K(+) matched the experimental results and suggest that d(TGGGGT)4 contains a 5'-capping cation in solution.

Torque generation by axonemal outer-arm dynein.

Outer-arm dynein is the main engine providing the motive force in cilia. Using three-dimensional tracking microscopy, we found that contrary to previous reports Tetrahymena ciliary three-headed outer-arm dynein (αßγ) as well as proteolytically generated two-headed (ßγ) and one-headed (α) subparticles showed clockwise rotation of each sliding microtubule around its longitudinal axis in microtubule corkscrewing assays. By measuring the rotational pitch as a function of ATP concentration, we also found that the microtubule corkscrewing pitch is independent of ATP concentration, except at low ATP concentrations where the pitch generated by both three-headed αßγ and one-headed α exhibited significantly longer pitch. In contrast, the pitch driven by two-headed ßγ did not display this sensitivity. In the assays on lawns containing mixtures of α and ßγ at various ratios, the corkscrewing pitch increased dramatically in a nonlinear fashion as the ratio of α in the mixture increased. Even small proportions of α-subparticle could significantly increase the corkscrewing pitch of the mixture. Our data show that torque generation does not require the three-headed outer-arm dynein (αßγ) but is an intrinsic property of the subparticles of axonemal dyneins and also suggest that each subparticle may have distinct mechanical properties.

RNA tertiary structure analysis by 2'-hydroxyl molecular interference.

We introduce a melded chemical and computational approach for probing and modeling higher-order intramolecular tertiary interactions in RNA. 2'-Hydroxyl molecular interference (HMX) identifies nucleotides in highly packed regions of an RNA by exploiting the ability of bulky adducts at the 2'-hydroxyl position to disrupt overall RNA structure. HMX was found to be exceptionally selective for quantitative detection of higher-order and tertiary interactions. When incorporated as experimental constraints in discrete molecular dynamics simulations, HMX information yielded accurate three-dimensional models, emphasizing the power of molecular interference to guide RNA tertiary structure analysis and fold refinement. In the case of a large, multidomain RNA, the Tetrahymena group I intron, HMX identified multiple distinct sets of tertiary structure interaction groups in a single, concise experiment.

Single-molecule correlated chemical probing of RNA.

Complex higher-order RNA structures play critical roles in all facets of gene expression; however, the through-space interaction networks that define tertiary structures and govern sampling of multiple conformations are poorly understood. Here we describe single-molecule RNA structure analysis in which multiple sites of chemical modification are identified in single RNA strands by massively parallel sequencing and then analyzed for correlated and clustered interactions. The strategy thus identifies RNA interaction groups by mutational profiling (RING-MaP) and makes possible two expansive applications. First, we identify through-space interactions, create 3D models for RNAs spanning 80-265 nucleotides, and characterize broad classes of intramolecular interactions that stabilize RNA. Second, we distinguish distinct conformations in solution ensembles and reveal previously undetected hidden states and large-scale structural reconfigurations that occur in unfolded RNAs relative to native states. RING-MaP single-molecule nucleic acid structure interrogation enables concise and facile analysis of the global architectures and multiple conformations that govern function in RNA.

Identification of biotin carboxyl carrier protein in Tetrahymena and its application in in vitro motility systems of outer arm dynein.

Axonemal dynein plays a central role in ciliary beating. Recently, a functional expression system of axonemal dynein was established in the ciliated protozoan Tetrahymena. This study identifies biotin carboxyl carrier protein (BCCP) in Tetrahymena and demonstrates its application in in vitro motility systems of outer arm dynein.

Folding pathways of the Tetrahymena ribozyme.

Like many structured RNAs, the Tetrahymena group I intron ribozyme folds through multiple pathways and intermediates. Under standard conditions in vitro, a small fraction reaches the native state (N) with kobs ≈ 0.6 min(-1), while the remainder forms a long-lived misfolded conformation (M) thought to differ in topology. These alternative outcomes reflect a pathway that branches late in folding, after disruption of a trapped intermediate (Itrap). Here we use catalytic activity to probe the folding transitions from Itrap to the native and misfolded states. We show that mutations predicted to weaken the core helix P3 do not increase the rate of folding from Itrap but they increase the fraction that reaches the native state rather than forming the misfolded state. Thus, P3 is disrupted during folding to the native state but not to the misfolded state, and P3 disruption occurs after the rate-limiting step. Interestingly, P3-strengthening mutants also increase native folding. Additional experiments show that these mutants are rapidly committed to folding to the native state, although they reach the native state with approximately the same rate constant as the wild-type ribozyme (~1 min(-1)). Thus, the P3-strengthening mutants populate a distinct pathway that includes at least one intermediate but avoids the M state, most likely because P3 and the correct topology are formed early. Our results highlight multiple pathways in RNA folding and illustrate how kinetic competitions between rapid events can have long-lasting effects because the "choice" is enforced by energy barriers that grow larger as folding progresses.

The functional expression and motile properties of recombinant outer arm dynein from Tetrahymena.

Cilia and flagella are motile organelles that play various roles in eukaryotic cells. Ciliary movement is driven by axonemal dyneins (outer arm and inner arm dyneins) that bind to peripheral microtubule doublets. Elucidating the molecular mechanism of ciliary movement requires the genetic engineering of axonemal dyneins; however, no expression system for axonemal dyneins has been previously established. This study is the first to purify recombinant axonemal dynein with motile activity. In the ciliated protozoan Tetrahymena, recombinant outer arm dynein purified from ciliary extract was able to slide microtubules in a gliding assay. Furthermore, the recombinant dynein moved processively along microtubules in a single-molecule motility assay. This expression system will be useful for investigating the unique properties of diverse axonemal dyneins and will enable future molecular studies on ciliary movement.

Roles of long-range tertiary interactions in limiting dynamics of the Tetrahymena group I ribozyme.

We determined the effects of mutating the long-range tertiary contacts of the Tetrahymena group I ribozyme on the dynamics of its substrate helix (referred to as P1) and on catalytic activity. Dynamics were assayed by fluorescence anisotropy of the fluorescent base analogue, 6-methyl isoxanthopterin, incorporated into the P1 helix, and fluorescence anisotropy and catalytic activity were measured for wild type and mutant ribozymes over a range of conditions. Remarkably, catalytic activity correlated with P1 anisotropy over 5 orders of magnitude of activity, with a correlation coefficient of 0.94. The functional and dynamic effects from simultaneous mutation of the two long-range contacts that weaken P1 docking are cumulative and, based on this RNA's topology, suggest distinct underlying origins for the mutant effects. Tests of mechanistic predictions via single molecule FRET measurements of rate constants for P1 docking and undocking suggest that ablation of the P14 tertiary interaction frees P2 and thereby enhances the conformational space explored by the undocked attached P1 helix. In contrast, mutation of the metal core tertiary interaction disrupts the conserved core into which the P1 helix docks. Thus, despite following a single correlation, the two long-range tertiary contacts facilitate P1 helix docking by distinct mechanisms. These results also demonstrate that a fluorescence anisotropy probe incorporated into a specific helix within a larger RNA can report on changes in local helical motions as well as differences in more global dynamics. This ability will help uncover the physical properties and behaviors that underlie the function of RNAs and RNA/protein complexes.

RNA catalytic activity as a probe of chaperone-mediated RNA folding.

For structured RNAs that possess catalytic activity, this activity provides a powerful probe for measuring the progress of folding and the effects of RNA chaperone proteins on the folding rate. The crux of this approach is that only the natively folded RNA is able to perform the catalytic reaction. This method can provide a quantitative measure of the fraction of native RNA over time, and it can readily distinguish the native state from all misfolded conformations. Here we describe an activity-based method measuring native folding of ribozymes derived from self-splicing group I introns, and we show how the assay can be used to monitor acceleration of native folding by DEAD-box RNA helicase proteins that function as general RNA chaperones. By measuring the amount of substrate that is converted to product in a rapid first turnover, we describe how to determine the fraction of the ribozyme population that is present in the native state. Further, we describe how to perform a two-stage or discontinuous assay in which folding proceeds in stage one and then solution conditions are changed in stage two to permit catalytic activity and block further folding. This protocol allows folding to be followed under a broad range of solution conditions, including those that do not support catalytic activity, and facilitates studies of chaperone proteins.

Role of ion valence in the submillisecond collapse and folding of a small RNA domain.

Following the addition of ions to trigger folding, RNA molecules undergo a transition from rigid, extended states to a compact ensemble. Determining the time scale for this collapse provides important insights into electrostatic contributions to RNA folding; however, it can be challenging to isolate the effects of purely nonspecific collapse, e.g., relaxation due to backbone charge compensation, from the concurrent formation of some tertiary contacts. To solve this problem, we decoupled nonspecific collapse from tertiary folding using a single-point mutation to eliminate tertiary contacts in the small RNA subdomain known as tP5abc. Microfluidic mixing with microsecond time resolution and Förster resonance energy transfer detection provides insight into the ionic strength-dependent transition from extended to compact ensembles. Differences in reaction rates are detected when folding is initiated by monovalent or divalent ions, consistent with equilibrium measurements illustrating the enhanced screening of divalent ions relative to monovalent ions at the same ionic strength. Ion-driven collapse is fast, and a comparison of the collapse time of the wild-type and mutant tP5abc suggests that site binding of Mg(2+) occurs on submillisecond time scales.

G-quadruplexes form ultrastable parallel structures in deep eutectic solvent.

G-quadruplex DNA is highly polymorphic. Its conformation transition is involved in a series of important life events. These controllable diverse structures also make G-quadruplex DNA a promising candidate as catalyst, biosensor, and DNA-based architecture. So far, G-quadruplex DNA-based applications are restricted done in aqueous media. Since many chemical reactions and devices are required to be performed under strictly anhydrous conditions, even at high temperature, it is challenging and meaningful to conduct G-quadruplex DNA in water-free medium. In this report, we systemically studied 10 representative G-quadruplexes in anhydrous room-temperature deep eutectic solvents (DESs). The results indicate that intramolecular, intermolecular, and even higher-order G-quadruplex structures can be formed in DES. Intriguingly, in DES, parallel structure becomes the G-quadruplex DNA preferred conformation. More importantly, compared to aqueous media, G-quadruplex has ultrastability in DES and, surprisingly, some G-quadruplex DNA can survive even beyond 110 °C. Our work would shed light on the applications of G-quadruplex DNA to chemical reactions and DNA-based devices performed in an anhydrous environment, even at high temperature.

Tetrahymena in the laboratory: strain resources, methods for culture, maintenance, and storage.

The ciliated protozoan Tetrahymena thermophila has been an important model system for biological research for many years. During that time, a variety of useful strains, including highly inbred stocks, a collection of diverse mutant strains, and wild cultivars from a variety of geographical locations have been identified. In addition, thanks to the efforts of many different laboratories, optimal conditions for growth, maintenance, and storage of Tetrahymena have been worked out. To facilitate the efficient use of Tetrahymena, especially by those new to the system, this chapter presents a brief description of many available Tetrahymena strains and lists possible resources for obtaining viable cultures of T. thermophila and other Tetrahymena species. Descriptions of commonly used media, methods for cell culture and maintenance, and protocols for short- and long-term storage are also presented.