Telomerase can act as a template- and RNA-independent terminal transferase

Lue, Neal F.; Bosoy, Dimitry; Moriarty, Tara J.; Autexier, Chantal; Altman, Brian J.; Leng, Siyang

doi:10.1073/pnas.0502252102

Cited by 47 publications

(31 citation statements)

References 52 publications

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“…S1C). It was important to ensure that calcium ions, required for CaM-CBP interactions, did not interfere with pol II elongation because such ions abolish transcription activity in a related system (23). Based on the unaltered function of the pol II elongation complexes, we believe that the labeling used in our experiments would not cause significant structural perturbations of the complexes.…”

Section: Resultsmentioning

confidence: 88%

Mapping RNA exit channel on transcribing RNA polymerase II by FRET analysis

Chen¹,

Chang²,

Yen³

et al. 2009

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

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A simple genetic tag-based labeling method that permits specific attachment of a fluorescence probe near the C terminus of virtually any subunit of a protein complex is implemented. Its immediate application to yeast RNA polymerase II (pol II) enables us to test various hypotheses of RNA exit channel by using fluorescence resonance energy transfer (FRET) analysis. The donor dye is labeled on a site near subunit Rpb3 or Rpb4, and the acceptor dye is attached to the 5 end of RNA transcript in the pol II elongation complex. Both in-gel and single-molecule FRET analysis show that the growing RNA is leading toward Rpb4, not Rpb3, supporting the notion that RNA exits through the proposed channel 1. Distance constraints derived from our FRET results, in conjunction with triangulation, reveal the exit track of RNA transcript on core pol II by identifying amino acids in the vicinity of the 5 end of RNA and show that the extending RNA forms contacts with the Rpb7 subunit. The significance of RNA exit route in promoter escape and that in cotranscriptional mRNA processing is discussed.nanometry ͉ structure ͉ transcription ͉ in-gel ͉ single-molecule fluorescence R NA polymerase II (pol II), a protein complex containing 12 subunits, Rpb1-Rpb12, of a total mass of Ϸ500 kDa and size Ϸ100-140 Å, is the enzyme machinery synthesizing mRNA in all eukaryotes (1). X-ray studies of pol II complexes (2-4) led to an atomic model containing structural elements with functional implications (Fig. 1A). In a transcribing pol II, between the ''clamp'' and ''jaw'' domain, lies a cleft (4) that harbors the active center, a straight duplex DNA and an RNA-DNA hybrid (position ϩ1 to Ϫ8, ϩ1 denoting the nucleotide addition site). The strand separation of RNA from DNA template occurs upstream of the hybrid at positions Ϫ9 and Ϫ10, facilitated by a set of protein loops including the ''lid'' domain as a driving wedge. Nascent RNA moves through an exit pore from the active center, crossing a saddle-like surface, beneath an ''arch'' bridging the clamp and wall (5).How does pol II instruct the nascent RNA to exit beyond the saddle? Is there a unique path on pol II connecting the active center to its exterior that nascent RNA may follow? To date, insights into the RNA exit have come from analysis of pol II surface charge distribution: two positively charged grooves, on either side of the ''dock domain'' (Fig. 1 A), can accommodate ssRNA (5). One groove, putatively referred as ''exit channel 1,'' runs around the base of the clamp, leading toward the stalk of subcomplex Rpb4-Rpb7, which can bind RNA via its ribonucleoprotein fold (6, 7). The other groove, termed ''exit channel 2,'' runs down the back side of pol II, through Rpb3 and Rpb11, leading toward Rpb8, a subunit equally competent in RNA binding by its single-strand nucleic acid-binding motif. Intriguingly, exit channel 1 would cause the RNA to bend sharply, implying that channel 2 is energetically favored for RNA binding. Yet, evidence in support of the channel 1 hypothesis has come from observation...

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Section: Resultsmentioning

confidence: 88%

Mapping RNA exit channel on transcribing RNA polymerase II by FRET analysis

Chen¹,

Chang²,

Yen³

et al. 2009

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

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“…Mn 2ϩ -stimulated transferase activity of template-dependent polymerases is not without precedence. For example, telomerase, another specialized RT that shares a number of interesting properties with hepadnavirus polymerases, including the presence of a specific RNA (the telomerase RNA [TR]) as an integral component of the enzyme complex and as the specific template, was recently shown to display a transferase-like activity that is independent of TR and is evident only in the presence of Mn 2ϩ (58). There are several novel aspects of our findings that differentiate our findings from previous reports of transferase activities.…”

Section: Figmentioning

confidence: 99%

Protein-Primed Terminal Transferase Activity of Hepatitis B Virus Polymerase

Jones

2013

J Virol

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Hepatitis B virus (HBV) replication requires reverse transcription of an RNA pregenome (pgRNA) by a multifunctional polymerase (HP). HP initiates viral DNA synthesis by using itself as a protein primer and an RNA signal on pgRNA, termed epsilon (H), as the obligatory template. We discovered a Mn 2؉ -dependent transferase activity of HP in vitro that was independent of H but also used HP as a protein primer. This protein-primed transferase activity was completely dependent on the HP polymerase active site. The DNA products of the transferase reaction were linked to HP via a phosphotyrosyl bond, and replacement of the Y63 residue of HP, the priming site for templated DNA synthesis, almost completely eliminated DNA synthesis by the transferase activity, suggesting that Y63 also serves as the predominant priming site for the transferase reaction. For this transferase activity, HP could use all four deoxynucleotide substrates, but TTP was clearly favored for extensive polymerization. The transferase activity was highly distributive, leading to the synthesis of DNA homo-and hetero-oligomeric and -polymeric ladders ranging from 1 nucleotide (nt) to >100 nt in length, with single-nt increments. As with H-templated DNA synthesis, the protein-primed transferase reaction was characterized by an initial stage that was resistant to the pyrophosphate analog phosphonoformic acid (PFA) followed by PFA-sensitive DNA synthesis, suggestive of an HP conformational change upon the synthesis of a nascent DNA oligomer. These findings have important implications for HBV replication, pathogenesis, and therapy. H epatitis B virus (HBV)is an important human pathogen and a member of the family Hepadnaviridae, which comprises a group of pararetroviruses replicating a double-stranded DNA genome through an RNA intermediate called pregenomic RNA (pgRNA) by a multifunctional viral polymerase protein (HP), a specialized reverse transcriptase (RT) (1-4). Structurally, HP is divided into four domains, including (from the N to the C terminus) the terminal protein (TP), spacer, RT, and RNase H domains (5-7). Although the RT and RNase H domains are conserved with those found in other RT enzymes, TP is unique to hepadnaviruses, and the spacer has no known function other than serving as a tether between the TP and RT domains (5, 7-11). As with other RTs, HP has RNA-and DNA-dependent DNA polymerase activity. Furthermore, HP also serves as a protein primer; specifically, a highly conserved Y residue in its TP domain (Y63) is used as a primer to initiate viral minus-strand DNA [(Ϫ)-DNA] synthesis (12, 13). This protein-primed reverse transcription requires a specific HBV RNA template called epsilon (Hε), located at the 5= end of the pgRNA, which is specifically recognized by HP and is thought to be an integral component of the HP holoenzyme (14-21). HP-Hε interactions trigger conformational changes in both the protein and RNA of the resulting ribonucleoprotein (RNP) complex, which are thought to be required functionally for HP to gain catalytic activity a...

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“…The ability of RVT to add NTPs and, to a lesser extent, dNTPs to 39-OH termini places it apart from conventional RTs and closer to TERTs, which are known to exhibit RdRP and template-independent terminal deoxynucleotidyltransferase (TdT) activity in addition to RT activity (24,25). The emergence of a unique type of RT-related genes raises questions about their relationship to other RT-like proteins.…”

Section: Resultsmentioning

confidence: 99%

A widespread class of reverse transcriptase-related cellular genes

Gladyshev

Arkhipova

2011

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

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Reverse transcriptases (RTs) polymerize DNA on RNA templates. They fall into several structurally related but distinct classes and form an assemblage of RT-like enzymes that, in addition to RTs, also includes certain viral RNA-dependent RNA polymerases (RdRP) synthesizing RNA on RNA templates. It is generally believed that most RT-like enzymes originate from retrotransposons or viruses and have no specific function in the host cell, with telomerases being the only notable exception. Here we report on the discovery and properties of a unique class of RT-related cellular genes collectively named rvt. We present evidence that rvts are not components of retrotransposons or viruses, but single-copy genes with a characteristic domain structure that may contain introns in evolutionarily conserved positions, occur in syntenic regions, and evolve under purifying selection. These genes can be found in all major taxonomic groups including protists, fungi, animals, plants, and even bacteria, although they exhibit patchy phylogenetic distribution in each kingdom. We also show that the RVT protein purified from one of its natural hosts, Neurospora crassa, exists in a multimeric form and has the ability to polymerize NTPs as well as dNTPs in vitro, with a strong preference for NTPs, using Mn 2+ as a cofactor. The existence of a previously unknown class of single-copy RT-related genes calls for reevaluation of the current views on evolution and functional roles of RNA-dependent polymerases in living cells.D NA-dependent polymerases are essential for cellular function, as they mediate the flow of genetic information from DNA to RNA to proteins (1). In contrast, RNA-dependent polymerases have long been associated with replication of selfish and parasitic genetic elements, such as viruses or transposons. Although the discovery of reverse transcriptase (RT) challenged the concept of unidirectionality of the flow of genetic information, this reverse direction has been reserved for retroviruses, pararetroviruses (hepadna-and caulimoviruses), and other RT-containing multicopy entities such as non-LTR and LTR retrotransposons, group II introns, retrons, and retroplasmids, as well as occasional retro (pseudo)genes (2-4). Similarly, viral RNA-dependent RNA polymerases (RdRPs), enzymes structurally related to RTs, serve to replicate the genomes of viruses that use RNA as genetic material (5). These and certain other polymerases are unified by the architecture known as "right hand," composed of the three subdomains called fingers, palm, and thumb (6). Like all polymerases, they use two-metal-ion catalysis for phosphoryl transfer reactions resulting in nucleotide addition.In 1997, this diverse superfamily of enzymes was joined by the telomerase reverse transcriptase (TERT), a specialized RT that maintains the ends of eukaryotic linear chromosomes by addition of short G-rich repeated DNA sequences that are copied multiple times via reverse transcription of a specific region of the associated RNA template constituting part of the holoenzyme (...

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Telomerase can act as a template- and RNA-independent terminal transferase

Cited by 47 publications

References 52 publications

Mapping RNA exit channel on transcribing RNA polymerase II by FRET analysis

Mapping RNA exit channel on transcribing RNA polymerase II by FRET analysis

Protein-Primed Terminal Transferase Activity of Hepatitis B Virus Polymerase

A widespread class of reverse transcriptase-related cellular genes

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