Structure of the Tribolium castaneum telomerase catalytic subunit TERT

Gillis, Andrew; Schuller, Anthony P.; Skordalakes, Emmanuel

doi:10.1038/nature07283

Cited by 250 publications

(305 citation statements)

References 55 publications

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“…S8 A and C). The remaining QFP may stabilize protein folding, as suggested by the Tribolium TERT structure (23). Motif CP is located opposite the CR4/5 binding pocket (Fig.…”

Section: Discussionmentioning

confidence: 95%

“…The ciliate TR lacks a three-way junction but contains the conserved stem-loop IV that binds TRBD and might function analogously to the vertebrate CR4/5 domain (16)(17)(18)(19)(20)(21). Although the structures of TRBD from Tetrahymena thermophila and Tribolium castaneum (red flour beetle) have been determined by X-ray crystallography (22,23), the details of the TR-TRBD binding interface remain elusive.…”

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confidence: 99%

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RNA–protein binding interface in the telomerase ribonucleoprotein

Bley¹,

Qi²,

Rand³

et al. 2011

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

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Telomerase is a specialized reverse transcriptase containing an intrinsic telomerase RNA (TR) which provides the template for telomeric DNA synthesis. Distinct from conventional reverse transcriptases, telomerase has evolved a unique TR-binding domain (TRBD) in the catalytic telomerase reverse transcriptase (TERT) protein, integral for ribonucleoprotein assembly. Two structural elements in the vertebrate TR, the pseudoknot and CR4/5, bind TERT independently and are essential for telomerase enzymatic activity. However, the details of the TR-TERT interaction have remained elusive. In this study, we employed a photoaffinity cross-linking approach to map the CR4/5-TRBD RNA-protein binding interface by identifying RNA and protein residues in close proximity. Photoreactive 5-iodouridines were incorporated into the medaka CR4/5 RNA fragment and UV cross-linked to the medaka TRBD protein fragment. The cross-linking RNA residues were identified by alkaline partial hydrolysis and cross-linked protein residues were identified by mass spectrometry. Three CR4/5 RNA residues (U182, U187, and U205) were found cross-linking to TRBD amino acids Tyr503, Phe355, and Trp477, respectively. This CR4/5 binding pocket is distinct and separate from the previously proposed T pocket in the Tetrahymena TRBD. Based on homologous structural models, our cross-linking data position the essential loop L6.1 adjacent to the TERT C-terminal extension domain. We thus propose that stem-loop 6.1 facilitates proper TERT folding by interacting with both TRBD and C-terminal extension. Revealing the telomerase CR4/5-TRBD binding interface with single-residue resolution provides important insights into telomerase ribonucleoprotein architecture and the function of the essential CR4/5 domain.

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“…S8 A and C). The remaining QFP may stabilize protein folding, as suggested by the Tribolium TERT structure (23). Motif CP is located opposite the CR4/5 binding pocket (Fig.…”

Section: Discussionmentioning

confidence: 95%

mentioning

confidence: 99%

RNA–protein binding interface in the telomerase ribonucleoprotein

Bley¹,

Qi²,

Rand³

et al. 2011

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

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“…Intriguingly, the catalytic domain of telomerase reverse transcriptase (TERT) has structural and functional characteristics in common with picornaviral RNA-dependent RNA polymerases. The catalytic domain of TERT assumes a right-hand conformation, with thumb, palm, and fingers domains encircling the telomerase RNA template and cDNA products (53)(54)(55). Furthermore, TERT uses a template-dependent reiterative transcription mechanism to synthesize repetitive DNA sequences of variable length at the 3= end of eukaryotic chromosomes (56).…”

Section: Discussionmentioning

confidence: 99%

Structural Features of a Picornavirus Polymerase Involved in the Polyadenylation of Viral RNA

Kempf

Kelly²,

Springer

et al. 2013

J Virol

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Picornaviruses have 3= polyadenylated RNA genomes, but the mechanisms by which these genomes are polyadenylated during viral replication remain obscure. Based on prior studies, we proposed a model wherein the poliovirus RNA-dependent RNA polymerase (3D pol ) uses a reiterative transcription mechanism while replicating the poly(A) and poly(U) portions of viral RNA templates. To further test this model, we examined whether mutations in 3D pol influenced the polyadenylation of virion RNA. We identified nine alanine substitution mutations in 3D pol that resulted in shorter or longer 3= poly(A) tails in virion RNA. These mutations could disrupt structural features of 3D pol required for the recruitment of a cellular poly(A) polymerase; however, the structural orientation of these residues suggests a direct role of 3D pol in the polyadenylation of RNA genomes. Reaction mixtures containing purified 3D pol and a template RNA with a defined poly(U) sequence provided data consistent with a template-dependent reiterative transcription mechanism for polyadenylation. The phylogenetically conserved structural features of 3D pol involved in the polyadenylation of virion RNA include a thumb domain alpha helix that is positioned in the minor groove of the double-stranded RNA product and lysine and arginine residues that interact with the phosphates of both the RNA template and product strands.P icornaviruses, like many other positive-strand RNA viruses, have RNA genomes with variable-length 3= poly(A) tails (1). The poly(A) tails of picornaviruses are important for viability (2), with the length of the poly(A) tails influencing the magnitudes of both viral mRNA translation and RNA replication (3, 4). RNA sequences and structures within the 3= nontranslated region are reported to influence both the length of poly(A) tails in picornaviral RNA (5) and the efficiency of virus replication (6, 7). Viral RNA-dependent RNA polymerases (3D pol s) are predicted to catalyze the polyadenylation of picornaviral RNA in a template-dependent manner during viral RNA replication (8); however, the mechanisms by which RNA genomes are polyadenylated during viral RNA replication remain obscure. In particular, there is little understanding of the mechanisms regulating the length of poly(A) tails synthesized during RNA replication. The RNA-dependent RNA polymerases of negative-strand RNA viruses reiteratively transcribe a small poly(U) sequence within intergenic regions of the viral RNA genome to produce long poly(A) tails on viral mRNAs (9). In a similar manner, the poliovirus RNA-dependent RNA polymerase can reiteratively transcribe poly(A) and poly(U) sequences during viral RNA replication, producing poly(A) and poly(U) sequences longer than those in the respective template RNAs (8).Viral RNA-dependent RNA polymerases are well studied at the structural level (10-12), yet there have been no reports describing the structural features of viral polymerases involved in the polyadenylation of viral RNA. The RNA-dependent RNA polymerase of picornaviruses a...

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“…A Model for TBE Interaction with the TERT RBD-Comparison of x-ray crystal structures of the T. castaneum TERT in complex with its DNA substrate and the T. thermophila RBD show significant structural homology (14,15,28). Alignment of the two structures reveals the likely position of the T. thermophila RBD with respect to the reverse transcriptase domains and the RNA template (Fig.…”

Section: Discussionmentioning

confidence: 99%

A Conserved Motif in Tetrahymena thermophila Telomerase Reverse Transcriptase Is Proximal to the RNA Template and Is Essential for Boundary Definition

Akiyama¹,

Gomez²,

Stone³

2013

Journal of Biological Chemistry

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Background: Telomerase requires the interaction of conserved protein and RNA motifs. Results: Site-directed hydroxyl radical probing and mutagenesis identified an essential region of RNA-protein interactions. Conclusion:The conserved CP2 protein motif is proximal to a conserved telomerase RNA motif. Significance: Understanding how telomerase protein and RNA subunits interact allows us to begin constructing a more complete mechanistic model of telomerase function.

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Structure of the Tribolium castaneum telomerase catalytic subunit TERT

Cited by 250 publications

References 55 publications

RNA–protein binding interface in the telomerase ribonucleoprotein

RNA–protein binding interface in the telomerase ribonucleoprotein

Structural Features of a Picornavirus Polymerase Involved in the Polyadenylation of Viral RNA

A Conserved Motif in Tetrahymena thermophila Telomerase Reverse Transcriptase Is Proximal to the RNA Template and Is Essential for Boundary Definition

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