Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.49 (reverse transcriptase)
31,746 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Telomerase is a reverse transcriptase that adds single-stranded telomeric repeats to the ends of linear eukaryotic chromosomes. It consists of an RNA molecule including a template sequence, a protein subunit containing reverse transcriptase motifs, and auxiliary proteins. We have carried out an interference footprinting analysis of the Tetrahymena telomerase elongation complexes. In this study, single-stranded oligonucleotide primers containing telomeric sequences were modified with base-specific chemical reagents and extended with the telomerase by a single (32)P-labeled dGMP or dTMP. Base modifications that interfered with the primer extension reactions were mapped by footprinting. Major functional interactions were detected between the telomerase and the six or seven 3'-terminal residues of the primers. These interactions occurred not only with the RNA template region, but also with another region in the enzyme ribonucleoprotein complex designated the telomerase DNA interacting surface (TDIS). This was indicated by footprints generated with dimethyl sulfate (that did not affect Watson-Crick hydrogen bonding) and by footprinting assays performed with mutant primers. In primers aligned at a distance of 2 nucleotides along the RNA template region, the footprints of the six or seven 3'-terminal residues were shifted by 2 nucleotides. This shift indicated that during the elongation reaction, TDIS moved in concert with the 3' ends of the primers relative to the template region. Weak interactions occurred between the telomerase and residues located upstream of the seventh nucleotide. These interactions were stronger in primers that were impaired in the ability to align with the template.
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PMID:Interference footprinting analysis of telomerase elongation complexes. 1082 87

Telomerase is a ribonucleoprotein reverse transcriptase responsible for the maintenance of one strand of telomere terminal repeats. The key protein subunit of the telomerase complex, known as TERT, possesses reverse transcriptase-like motifs that presumably mediate catalysis. These motifs are located in the C-terminal region of the polypeptide. Hidden Markov model-based sequence analysis revealed in the N-terminal region of all TERTs the presence of four conserved motifs, named GQ, CP, QFP, and T. Point mutation analysis of conserved residues confirmed the functional importance of the GQ motif. In addition, the distinct phenotypes of the GQ mutants suggest that this motif may play at least two distinct functions in telomere maintenance. Deletion analysis indicates that even the most N-terminal nonconserved region of yeast TERT (N region) is required for telomerase function. This N region exhibits a nonspecific nucleic acid binding activity that probably reflects an important physiologic function. Expression studies of various portions of the yeast TERT in Escherichia coli suggest that the N region and the GQ motif together may constitute a stable domain. We propose that all TERTs may have a bipartite organization, with an N-GQ domain connected to the other motifs through a flexible linker.
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PMID:Identification of functionally important domains in the N-terminal region of telomerase reverse transcriptase. 1086 75

Telomerase is a cellular reverse transcriptase specialized for use of a template carried within the RNA component of the enzyme ribonucleoprotein complex. Substrates for telomerase are single-stranded oligonucleotides in vitro and chromosome ends in vivo. In vitro, a bound substrate is extended by an initial round of DNA synthesis on the internal RNA template and in some cases by multiple rounds of template copying before product dissociation. In vivo, de novo synthesis of one strand of a telomeric repeat sequence by telomerase balances the sequence loss resulting from incomplete replication of linear chromosome ends by RNA primer-requiring DNA polymerases. Telomerase biochemistry has been studied extensively by using partially purified cell extracts. Telomerase components are being identified and beginning to be produced in recombinant form. This review focuses on the enzyme mechanism of telomerases from ciliate species, thus far the most intensively studied systems.
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PMID:Ciliate telomerase biochemistry. 1087 48

The ribonucleoprotein enzyme telomerase extends chromosome ends by copying a specific template sequence within its integral RNA component. An active recombinant telomerase RNP is minimally composed of this RNA and the telomerase reverse transcriptase (TERT) protein, which contains sequence motifs conserved among viral reverse transcriptases (RTs), flanked by N- and C-terminal extensions specific to TERTs. We have used site-directed mutagenesis to explore the roles of Tetrahymena TERT in determining features of telomerase activity in general and in establishing the boundaries and use of an internal RNA template in specific. We identify a new ciliate-specific motif in the TERT N-terminus required for template definition. Moreover, several residues in reverse transcriptase motifs 1, 2, A and D are critical for specific aspects of internal template use. Our results indicate that the unique specificity of telomerase activity is conferred to a reverse transcriptase active site by TERT residues both within and beyond the RT motif region.
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PMID:Template definition by Tetrahymena telomerase reverse transcriptase. 1094 24

The hepadnavirus reverse transcriptase binds cotranslationally to the viral pregenomic RNA. This ribonucleoprotein complex is then encapsidated into nascent viral core particles, where the reverse transcriptase copies the viral RNA into DNA. Here we report that 75% of the duck hepatitis B virus reverse transcriptase present in transfected LMH cells does not follow this well-known pathway but rather exists in the cell separate from the core protein or nucleocapsids. The nonencapsidated reverse transcriptase is also abundant in infected duck liver. The nonencapsidated reverse transcriptase exists as a complex set of isoforms that are most likely produced by posttranslational modification. Interestingly, only the smallest of these isoforms is encapsidated into viral core particles. The nonencapsidated reverse transcriptase is bound to a large cellular cytoplasmic structure(s) in a detergent-sensitive complex. The cellular distribution of the reverse transcriptase only partially overlaps that of the core protein, and this distribution is unaffected by blocking encapsidation. These observations raise the possibilities that the metabolic fate of the reverse transcriptase may be posttranscriptionally regulated and that the reverse transcriptase may have roles in the viral replication cycle beyond its well-known function in copying the viral genome.
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PMID:The majority of duck hepatitis B virus reverse transcriptase in cells is nonencapsidated and is bound to a cytoplasmic structure. 1095 66

Telomerase is a ribonucleoprotein that mediates extension of the dG-rich strand of telomeres in most eukaryotes. Like telomerase derived from ciliated protozoa, yeast telomerase is found to possess a tightly associated endonuclease activity that copurifies with the polymerization activity over different affinity-chromatographic steps. As is the case for ciliate telomerase, primers containing sequences that are not complementary to the RNA template can be efficiently cleaved by the yeast enzyme. More interestingly, we found that for the yeast enzyme, cleavage site selection is not stringent, since blocking cleavage at one site by the introduction of a nonhydrolyzable linkage can lead to the utilization of other sites. In addition, the reverse transcriptase activity of yeast telomerase can extend either the 5'- or 3'-end fragment following cleavage. Two general models that are consistent with the biochemical properties of the enzyme are presented: one model postulates two distinct active sites for the nuclease and reverse transcriptase, and the other invokes a multimeric enzyme with each protomer containing a single active site capable of mediating both cleavage and extension.
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PMID:Characterization of the interaction between the nuclease and reverse transcriptase activity of the yeast telomerase complex. 1095 77

Inhibition or activation of the reverse transcriptase telomerase can profoundly affect the proliferative capacity of normal cells and cancers. Here, we elucidate structural requirements for function of the essential RNA component of human telomerase, hTR. Two motifs within the independently stable H/ACA domain of hTR are required for accumulation of the mature RNA in vivo. However, these motifs can be substituted by a heterologous H/ACA family RNA. Two additional hTR elements are required both in vivo and in vitro for telomerase catalytic activity. Surprisingly, each of these elements independently binds to the telomerase reverse transcriptase. Our results establish fundamental differences between vertebrate and ciliate telomerase ribonucleoprotein architectures and also suggest strategies for the pharmaceutical development of telomerase-based anticancer therapies.
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PMID:Human telomerase activation requires two independent interactions between telomerase RNA and telomerase reverse transcriptase. 1098 83

Telomerase reverse transcriptase (TERT) differs from many other reverse transcriptases in that it remains stably associated with its template-containing RNA subunit. Elements of TERT involved in binding the RNA subunit have now been identified by mutagenesis and in vitro reconstitution of the Tetrahymena ribonucleoprotein complex. Mutations in the reverse transcriptase motifs of TERT reduced activity as expected but did not greatly reduce its binding to the telomerase RNA. In contrast, all mutations in the T and CP motifs dramatically reduced RNA binding. We therefore suggest that the T and CP motifs of TERT function to hold on to the telomerase RNA, leaving the RNA template region free to translocate through the RT domain.
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PMID:Telomerase RNA bound by protein motifs specific to telomerase reverse transcriptase. 1098 95

Reverse transcription in hepatitis B viruses is initiated through a unique protein priming mechanism whereby the viral reverse transcriptase (RT) first assembles into a ribonucleoprotein (RNP) complex with its RNA template and then initiates DNA synthesis de novo using the RT itself as a protein primer. RNP formation and protein priming require the assistance of host cell factors, including the molecular chaperone heat shock protein 90 (Hsp90). To better understand the mechanism of RT activation by Hsp90, we have now mapped the minimal RT sequences of the duck hepatitis B virus that are required for chaperone binding, RNP formation, and protein priming. Furthermore, we have reconstituted in vitro both RNP formation and protein priming using purified RT proteins and host factors. Our results show that (i) Hsp90 recognizes two independent domains of the RT, both of which are necessary for RNP formation and protein priming; (ii) Hsp90 function is required not only to establish, but also to maintain, the RT in a state competent for RNA binding; and (iii) Hsp90 is not required during RT synthesis and can activate the RT posttranslationally. Based on these findings, we propose a model for Hsp90 function whereby the chaperone acts as an active interdomain bridge to bring the two RT domains into a poised but labile conformation competent for RNP formation. It is anticipated that the reconstitution system established here will facilitate the isolation of additional host factors required for RT functions and further elucidation of the mechanisms of RT activation.
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PMID:In vitro reconstitution of a functional duck hepatitis B virus reverse transcriptase: posttranslational activation by Hsp90. 1109 Jan 40

Telomerase is a reverse transcriptase responsible for adding simple sequence repeats to chromosome 3'-ends. The template for telomeric repeat synthesis is carried within the RNA component of the telomerase ribonucleoprotein complex. Telomerases can copy their internal templates with repeat addition processivity, reusing the same template multiple times in the extension of a single primer. For some telomerases, optimal repeat addition processivity requires high micromolar dGTP concentrations, a much higher dGTP concentration than required for processive nucleotide addition within a repeat. We have investigated the requirements for dGTP-dependent repeat addition processivity using recombinant Tetrahymena telomerase. By altering the template sequence, we show that repeat addition processivity retains the same dGTP-dependence even if dGTP is not the first nucleotide incorporated in the second repeat. Furthermore, no dNTP other than dGTP can stimulate repeat addition processivity, even if it is the first nucleotide incorporated in the second repeat. Using structural variants of dGTP, we demonstrate that the stimulation of repeat addition processivity is specific for dGTP base and sugar constituents but requires only a single phosphate group. However, all nucleotides that stimulate repeat addition processivity also inhibit or compete with dGTP incorporation into product DNA. By assaying telomerase complexes reconstituted with a variety of altered templates, we find that repeat addition processivity has an unanticipated template or product sequence specificity. Finally, we show that a novel, nascent product DNA binding site establishes dGTP-dependent repeat addition processivity.
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PMID:Requirements for the dGTP-dependent repeat addition processivity of recombinant Tetrahymena telomerase. 1109 70


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