Tetrahymena telomerase ribonucleoprotein RNA-protein interactions

Telomerase is an enzyme that is essential for the replication and maintenance of chromosomal termini. It is a ribonucleoprotein consisting of a catalytic subunit, one or more associated proteins, and an integral RNA subunit that serves as a template for the synthesisof telomeric repeats. We identified a Tetrahymena telomerase RNA-protein complex by an electrophoretic mobility shift assay, using telomerase partially purified from whole cell extracts and radiolabeled, in vitro transcribed wild-type Tetrahymena telomerase RNA. Complex formation was specific as unlabeled Tetra-hymena telomerase RNA, but not Escherichia coli ribo-somal RNAs, competitively inhibited complex formation. Binding required concentrations of MgCl2of at least 10 mM and occurred over a wide range of potassium glutamate concentrations (20-220 mM). The RNA-protein complex was optimally reconstituted with a 30 degrees C preincubation for 相似文献   

Developmentally programmed healing of chromosomes by telomerase in Tetrahymena

Healing of a broken chromosome and in eukaryotes involves acquisition of a telomere. During macronuclear development in ciliated protozoans, germline chromosomes are fragmented into linear subchromosomes, whose ends are healed by de novo addition of telomeres. We showed previously that the ribonucleoprotein enzyme telomerase elongates preexisting telomeres by synthesizing one telomeric DNA strand, using a template sequence in the RNA moiety of the enzyme. By marking telomerase with a mutation in the telomerase RNA template, which causes synthesis of novel telomeric sequences, we now show that in the ciliate Tetrahymena, telomerase directly adds telomeric DNA onto nontelomeric sequences during developmentally controlled chromosome healing. Unexpectedly, one telomerase RNA template mutation converted telomerase from an enzyme that normally synthesizes precisely templated sequences to a less precise polymerase that sometimes synthesizes irregular telomeric repeats in vivo.     

De novo telomere addition by Tetrahymena telomerase in vitro.

Previous molecular genetic studies have shown that during programmed chromosomal healing, telomerase adds telomeric repeats directly to non-telomeric sequences in Tetrahymena, forming de novo telomeres. However, the biochemical mechanism underlying this process is not well understood. Here, we show for the first time that telomerase activity is capable in vitro of efficiently elongating completely non-telomeric DNA oligonucleotide primers, consisting of natural telomere-adjacent or random sequences, at low primer concentrations. Telomerase activity isolated from mated or vegetative cells had indistinguishable specificities for nontelomeric and telomeric primers. Consistent with in vivo results, the sequence GGGGT... was the predominant initial DNA sequence added by telomerase in vitro onto the 3' end of the non-telomeric primers. The 3' and 5' sequences of the primer both influenced the efficiency and pattern of de novo telomeric DNA addition. Priming of telomerase by double-stranded primers with overhangs of various lengths showed a requirement for a minimal 3' overhang of 20 nucleotides. With fully single-stranded non-telomeric primers, primer length up to approximately 30 nucleotides strongly affected the efficiency of telomeric DNA addition. We propose a model for the primer binding site of telomerase for non-telomeric primers to account for these length and structural requirements. We also propose that programmed de novo telomere addition in vivo is achieved through a hitherto undetected intrinsic ability of telomerase to elongate completely non-telomeric sequences.     

Tetrahymena telomerase RNA levels increase during macronuclear development.

Telomeres, the G-rich sequences found at the ends of eukaryotic chromosomes, ensure chromosome stability and prevent sequence loss from chromosome ends during DNA replication. During macronuclear development in Tetrahymena, the chromosomes fragment into pieces ranging from 20 kb to 1,500 kb. Tetrahymena telomerase, a ribonucleoprotein, adds telomeric (TTGGGG)n repeats onto telomeres and onto the newly generated macronuclear DNA ends. We have investigated whether telomerase RNA levels increase during macronuclear development, since such an increase might be expected during chromosomal fragmentation. The steady-state level of the telomerase RNA component was used to estimate the abundance of telomerase present in mating and nonmating Tetrahymena. Northern blot analysis revealed that in vegetatively growing Tetrahymena, there were 18,000-40,000 copies of telomerase RNA per cell. In mating cultures, the levels of RNA increased 2- to 5-fold at 9-15 h, and 1.5- to 3.5-fold in starved nonmating cultures. This increase in telomerase RNA paralleled telomerase activity, which also increased slightly in mating and starved nonmating cells.     

Using electrostatic potentials to predict DNA-binding sites on DNA-binding proteins

A method to detect DNA-binding sites on the surface of a protein structure is important for functional annotation. This work describes the analysis of residue patches on the surface of DNA-binding proteins and the development of a method of predicting DNA-binding sites using a single feature of these surface patches. Surface patches and the DNA-binding sites were initially analysed for accessibility, electrostatic potential, residue propensity, hydrophobicity and residue conservation. From this, it was observed that the DNA-binding sites were, in general, amongst the top 10% of patches with the largest positive electrostatic scores. This knowledge led to the development of a prediction method in which patches of surface residues were selected such that they excluded residues with negative electrostatic scores. This method was used to make predictions for a data set of 56 non-homologous DNA-binding proteins. Correct predictions made for 68% of the data set.     

Structural and functional insights into CST tethering in Tetrahymena thermophila telomerase

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Tracing the path of DNA substrates in active Tetrahymena telomerase holoenzyme complexes: mapping of DNA contact sites in the RNA subunit

Telomerase, the enzyme that extends single-stranded telomeric DNA, consists of an RNA subunit (TER) including a short template sequence, a catalytic protein (TERT) and accessory proteins. We used site-specific UV cross-linking to map the binding sites for DNA primers in TER within active Tetrahymena telomerase holoenzyme complexes. The mapping was performed at single-nucleotide resolution by a novel technique based on RNase H digestion of RNA-DNA hybrids made with overlapping complementary oligodeoxynucleotides. These data allowed tracing of the DNA path through the telomerase complexes from the template to the TERT binding element (TBE) region of TER. TBE is known to bind TERT and to be involved in the template 5'-boundary definition. Based on these findings, we propose that upstream sequences of each growing telomeric DNA chain are involved in regulation of its growth arrest at the 5'-end of the RNA template. The upstream DNA-TBE interaction may also function as an anchor for the subsequent realignment of the 3'-end of the DNA with the 3'-end of the template to enable initiation of synthesis of a new telomeric repeat.     

Multiple levels of telomerase regulation

Normally, cell division leads to shortening of telomeres, the nucleoprotein complexes located at the ends of linear chromosomes. When telomeres reach a critically short length, cells cease to divide. However, immortal tumor cells display stable telomere lengths and are able to maintain their proliferative state. Wong and colleagues have found that telomerase is sequestered by nucleoli during certain stages of the cell cycle, decreasing the likelihood of telomerase access to chromatin until the late S phase. Additionally, they demonstrate that ionizing radiation tends to keep telomerase sequestered in nucleoli, whereas cell transformation leads to telomerase translocation into the nucleoplasm, where, presumably, it can catalyze the lengthening of telomeres at appropriate and inappropriate sites. The sequestration of telomerase thus imposes a newly identified level of regulation on telomerase activity, implicating telomerase localization as a potentially useful target for pharmacotherapy.     

Mutational analysis of the Tetrahymena telomerase RNA: identification of residues affecting telomerase activity in vitro.

Telomere-specific repeat sequences are essential for chromosome end stability. Telomerase maintains telomere length by adding sequences de novo onto chromosome ends. The template domain of the telomerase RNA component dictates synthesis of species-specific telomeric repeats and other regions of the RNA have been suggested to be important for enzyme structure and/or catalysis. Using enzyme reconstituted in vitro with RNAs containing deletions or substitutions we identified nucleotides in the RNA component that are important for telomerase activity. Although many changes to conserved features in the RNA secondary structure did not abolish enzyme activity, levels of activity were often greatly reduced, suggesting that regions other than the template play a role in telomerase function. The template boundary was only altered by changes in stem II that affected the conserved region upstream of the template, not by changes in other regions, such as stems I, III and IV, consistent with a role of the conserved region in defining the 5' boundary of the template. Surprisingly, telomerase RNAs with substitutions or deletion of residues potentially abolishing the conserved pseudoknot structure had wild-type levels of telomerase activity. This suggests that this base pairing interaction may not be required for telomerase activity per se but may be conserved as a regulatory site for the enzyme in vivo.     

Utilization of ribonucleotides and RNA primers by Tetrahymena telomerase.

Telomerase is a ribonucleoprotein (RNP) DNA polymerase involved in telomere synthesis. A short sequence within the telomerase RNA component provides a template for de novo addition of the G-rich strand of a telomeric simple sequence repeat onto chromosome termini. In vitro, telomerase can elongate single-stranded DNA primers processively: one primer can be extended by multiple rounds of template copying before product dissociation. Telomerase will incorporate dNTPs or ddNTPs and will elongate any G-rich, single-stranded primer DNA. In this report, we show that Tetrahymena telomerase was able to incorporate a ribonucleotide, rGTP, into product polynucleotide. Synthesis of the product [d(TT)r(GGGG)]n was processive, suggesting that the chimeric product remained associated with the enzyme both at the active site and at a second, previously characterized, template-independent product binding site. As predicted by this finding, RNA-containing oligonucleotides served as primers for elongation. More than 3 nt of RNA at a primer 3' end decreased the quantity of product synthesis but increased the affinity of the primer for telomerase. Thus, RNA-containing primers were effective as competitive inhibitors of DNA primer elongation by telomerase. These results support the possible evolutionary origin of telomerase as an RNA-dependent RNA polymerase.