articles
Structural basis for telomerase catalytic subunit TERT
binding to RNA template and telomeric DNA
© 2010 Nature America, Inc. All rights reserved.
Meghan Mitchell1, Andrew Gillis1, Mizuko Futahashi2, Haruhiko Fujiwara2 & Emmanuel Skordalakes1
Telomerase is a specialized DNA polymerase that extends the 3′ ends of eukaryotic linear chromosomes, a process required for
genomic stability and cell viability. Here we present the crystal structure of the active Tribolium castaneum telomerase catalytic
subunit, TERT, bound to an RNA-DNA hairpin designed to resemble the putative RNA-templating region and telomeric DNA.
The RNA-DNA hybrid adopts a helical structure, docked in the interior cavity of the TERT ring. Contacts between the RNA
template and motifs 2 and B′ position the solvent-accessible RNA bases close to the enzyme active site for nucleotide binding and
selectivity. Nucleic acid binding induces rigid TERT conformational changes to form a tight catalytic complex. Overall, TERT–RNA
template and TERT–telomeric DNA associations are remarkably similar to those observed for retroviral reverse transcriptases,
suggesting common mechanistic aspects of DNA replication between the two families of enzymes.
Telomerase is a specialized ribonucleoprotein (RNP) reverse transcriptase1 that extends the 3′ ends of linear chromosomes2, which
would otherwise diminish in size with every cell division, eventually
leading to cellular aging3. Although this process is essential for cell
viability in healthy tissues early in life, the enzyme is switched off in
mature cells, ensuring a limited number of mitotic events. Tumors,
however, regain the ability to activate telomerase, which in turn
promotes the immortalization and spread of rapidly dividing ­cancer
cells4. Understanding the function of this enzyme at a molecular
level will aid in efforts to design therapies to combat both cancer and
age-related diseases3,5–7.
The catalytic subunit of telomerase is structurally similar to retro­
viral reverse transcriptases, viral RNA polymerases and, to a lesser
extent, the bacteriophage B-family DNA polymerases1. Like its structural homologs, the core catalytic subunit of telomerase, TERT, contains the fingers, palm and thumb domains1 required for nucleic acid
and nucleotide associations as well as catalysis. However, unlike other
polymerases, telomerase exists as a stable RNP complex and contains
unique domains, such as the universally conserved RNA binding
domain (TRBD), which is essential for RNP assembly8–10 and repeat
addition processivity11,12. A fifth domain, known as the TEN or the
anchor domain of telomerase, is present in several eukaryotes and has
been shown to be involved in single-stranded telomeric DNA binding13,14 and processivity15–17 in these organisms. In contrast to the
largely conserved protein subunit, the RNA component of telomerase
varies considerably in both size and sequence across species. Despite
these differences, telomerase RNAs contain conserved motifs18,19 (such
as the RNA-templating region essential for nucleotide binding and
selectivity), evidence that supports the existence of common mechanistic aspects of telomere replication across phylogenetic groups.
Telomerase is a high-fidelity enzyme in that it is able to add multiple
identical repeats of DNA, a capability that is conferred in large part by
its integral RNA-templating region (usually one-and-a-half telo­meric
repeats)20–24. Initiation of telomere replication leads to ­pairing of
the RNA-templating region with the incoming single-stranded DNA
primer to be extended20–22,24,25. Current evidence suggests that initial
RNA-DNA pairing occurs between 4 or 5 bases out of the possible
7–9 bases of the templating region, thus leaving a short string of RNA
nucleotides to be replicated before the 5′ end of the templating region
is reached20,26. Completion of one round of telomere extension leads
to a transient dissociation of the RNA-DNA hybrid, followed by repositioning of the DNA end at the active site of TERT and RNA-DNA
pairing at the other end of the template. Telomerase remains associated with the chromosome end until several telomeric repeats have
been added, a process that is highly regulated by telomere binding
proteins, such as the TPIP1–POT1 complex in humans27,28.
The structure of the T. castaneum full-length catalytic subunit of
telo­merase, TERT, together with existing biochemical data has provided significant insight into the mechanism of telomere replication by
telomerase. However, the precise mechanism of RNA-template usage
and DNA binding and positioning at the active site of the enzyme for
nucleotide incorporation are not clear. To address these questions, we
determined the high-resolution structure of the T. castaneum TERT
bound to an RNA-DNA hairpin designed to resemble its RNA template and the complementary DNA sequence. The structure revealed
that RNA-template binding by TERT is mediated by the conserved
motifs 2 and B′ of the fingers and palm domains, respectively, both
of which localize adjacent to and above the active site of the enzyme.
Contacts between the protein and the backbone of the RNA template position the solvent-accessible bases in proximity to the active
1Gene
Expression and Regulation Program, The Wistar Institute, Philadelphia, Pennsylvania, USA. 2Department of Integrated Biosciences, Graduate School of Frontier
Sciences, University of Tokyo, Kashiwa, Japan. Correspondence should be addressed to E.S. ([email protected]).
Received 17 October 2009; accepted 20 January 2010; published online 28 March 2010; doi:10.1038/nsmb.1777
nature structural & molecular biology VOLUME 17 NUMBER 4 APRIL 2010
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a
CP
T
TRBD
12
A IFD B′ C D E
Fingers
Palm
Thumb
loop helix
c
Thumb
RNA
templating
region
b
5′
3′
d
DNA
primer
5′
3′
e
Thumb
Palm
90°
DNA
© 2010 Nature America, Inc. All rights reserved.
TRBD
RNA
Fingers
site of the enzyme for nucleotide binding, thus facilitating ­selectivity.
TERT-DNA associations are largely mediated by the thumb loop and
helix. These contacts provide the stability required for a functional
telomerase elongation complex and facilitate positioning of the
3′-end nucleotides in proximity to the primer-grip region (motif E),
which in turn positions the 3′-end hydroxyl of the DNA primer at the
active site of the enzyme for nucleotide addition. Collectively, the data
presented here highlight the detailed interactions between TERT, the
RNA-templating region and the DNA primer to be extended during
the elongation process, providing novel insights into the mechanism
of telomere replication by telomerase.
RESULTS
TERT structure overview
We cocrystallized the full-length (Fig. 1a) active T. castaneum TERT
(Supplementary Fig. 1a,b) with an RNA-DNA hairpin containing the
putative RNA-templating region (5′-rCrUrGrArCrCrU-3′) and the
complementary telomeric DNA (5′-GTCAGGT-3′) joined together
with a short RNA-DNA linker (Fig. 1b and Supplementary Fig. 2).
Notably, the T. castaneum TERT lacks the TEN domain required for
activity and processivity in several eukaryotic telomerase genes, including those of humans17 and Tetrahymena thermophila29,30. The absence
of the TEN domain from T. castaneum could explain the reduced
activity observed for this enzyme when compared to those containing this domain. The RNA component of T. castaneum telo­merase
is not known, so we used the following information to predict its
templating region. The RNA-templating region of telomerase is usually one-and-a-half telomeric repeats22–24, and this sequence is known
for many organisms (see telomerase database at http://telomerase.asu.
edu/). For example, the mammalian telomerase-templating region is
‘CUAACCCU’, and the telomeric repeat is ‘TTAGGG’. The telomeric
repeat for T. castaneum is ‘TCAGG’31,32.
We designed the RNA-DNA hairpin to contain a three-nucleotide
overhang at the 5′ end of the RNA template so that we could trap the
enzyme in its catalytic state by cocrystallizing the protein–nucleic
acid assembly with Mg2+ ions and nonhydrolyzable nucleotides. In
an effort to identify a hairpin suitable for crystallographic studies,
we screened a number of RNA-DNA hairpins (15-mer, 18-mer and
21-mer; Supplementary Fig. 2) in which we kept the templating
region and the complementary DNA sequence the same but changed
the length of the linker. Although all three hairpins tested had the
same binding affinity for TERT and the same reverse transcriptase
514
Figure 1 Structure of the T. castaneum TERT–nucleic acid complex.
(a) Primary structure of the T. castaneum TERT. The TRBD, fingers, palm
and thumb domains are shown in blue, orange, tan and red, respectively.
Conserved motifs are also shown. (b) Primary structure of the RNA-DNA
hairpin (21-mer) cocrystallized with TERT. Blue, nucleic acid linker.
(c) Simulated annealed omit map of the RNA (magenta stick) and the DNA
(yellow stick) substrates at 1.0s contour level. (d) Cartoon representation of
the TERT–RNA-DNA complex; colors same as in a and c. (e) TERT surface
charge representation, showing the RNA-DNA hybrid (stick) docked in the
highly positively charged (blue color) cavity of the ring.
activity (data not shown), only the 21-mer was amenable to crystallo­
graphic studies. Notably, in the structure, the hairpin linker extends
out of the TERT ring and is only involved in crystal contacts with
adjacent molecules. We also grew the crystals in the presence of the
slowly hydrolyzable nucleotide analogs dNTPαS and Mg2+ ions.
We solved the structure at 2.7-Å resolution using the method
of molecular replacement, with the substrate-free TERT 1 (PDB
3DU6) as a search model (Table 1). All of the TERT molecule and
the RNA-DNA hybrid were interpretable in electron density maps.
Unexpectedly, there was extra density for three nucleotides at the
3′ end of the telomeric DNA (Fig. 1c), suggesting that TERT had
extended the 3′ end of the DNA substrate in the crystallization drop.
There was no evidence for nucleotide at the active site of the enzyme,
which was partially occupied by the nucleotide located at the 3′ end
of the DNA substrate.
Several lines of evidence indicate that the association of the
RNA-templating region and the DNA substrate with TERT in the
structure presented here are biologically relevant. First, TERT is an
active polymerase in the presence of the nucleic acid substrate used
in this study, as determined in standard reverse ­transcriptase assays
Table 1 Data collection and refinement statistics
TERT–RNA-DNA complex
Data collection
Space group
P 21
Cell dimensions
a, b, c (Å)
77.2, 52.8, 101.6
a, b, g (°)
90, 101.9, 90
Resolution (Å)
20–2.7 (2.85–2.70)
Rsym
11.4 (45.8)
I / σI
7.9 (2.3)
Completeness (%)
93.7 (96.1)
Redundancy
2.8 (2.5)
Refinement
Resolution (Å)
20–2.7
No. reflections
19,815
Rwork / Rfree
24.2 / 28.7
No. atoms
Protein
4,982
Ligand/ion
496/1
Water
54
B-factors
Protein
45
Ligand/ion
37
Water
21
R.m.s. deviations
Bond lengths (Å)
0.006
Bond angles (°)
0.887
One crystal was used. Values in parentheses are for highest-resolution shell.
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a
RNA
DNA
α10
b
α14
RNA
rA4
Palm
Active
site
5′
α13
Motif 2
Motif B′
Motif IFD
Fingers
α15
rU2
rC1
5′
Motif
T
Wat18
rG3
α15
Pro311
Motif
B′
Gly309
Val197
Ile196
Motif
2
© 2010 Nature America, Inc. All rights reserved.
Figure 2 TERT–RNA template associations. (a) RNA template (magenta
sticks) binding in the interior of the TERT ring is mediated by motifs 2
(cyan), B′ (purple) and helix α15 via a water molecule (Wat18). Contacts
between the RNA template and the protein place its 5′ end at the entry
of the RNA binding pocket (motif T, blue) of TRBD. (b) The nucleotide
located at the 5′ end of the RNA template (rC1) is coordinated by Ile196
and Val197 of motif 2 and Gly309 of motif B′. rU2 interacts with Pro311
of motif B′, and rG3 coordinates the backbone of helix α15 via a water
molecule (Wat18; red sphere).
(Supplementary Fig. 3) and in the crystallization drop. Second,
the RNA template makes contacts with conserved motifs that are
hallmarks of this family of enzymes. Third, contacts between TERT
and the RNA template position the solvent-accessible bases adjacent
to and above the active site of the enzyme for nucleotide binding,
thus facilitating selectivity. Moreover, TERT-RNA associations position the 5′ end of the RNA template at the entry of the TRBD RNA
binding pocket and where the template boundary element located
upstream of the RNA-template in many organisms is thought to
bind. Fourth, interactions between the DNA substrate and the
primer-grip region (a characteristic shared among telomerase and
HIV reverse transcriptases (HIV RTs)) place the 3′ end of the DNA
substrate at the active site of the enzyme, where it is accessible
for nucleotide addition. Fifth, TERT–nucleic acid associations are
strikingly similar to those observed for HIV RT, TERT’s closest
structural homolog.
The four major TERT domains—the RNA binding domain
(TRBD); the fingers domain, implicated in nucleotide binding and
processivity1,33; the palm domain, which contains the active site of
the enzyme1; and the thumb domain, implicated in DNA binding and
processivity1,34—are organized into a ring configuration similar to
that observed for the substrate-free enzyme1 (Fig. 1d). The arrangement of the TERT domains creates a highly positively charged cavity
in the interior of the TERT ring (Fig. 1e), which is 22 Å wide and
21 Å deep and can accommodate seven bases of double-stranded
nucleic acid. Within this cavity binds one molecule of the RNA-DNA
hybrid (Fig. 1d,e), which assembles via Watson-Crick base pairing
into a helical structure similar to both the DNA-DNA and RNA-DNA
structures bound to HIV RT35,36.
TERT nucleic acid associations
Interactions between the protein and the RNA-templating region are
mediated by the fingers, palm and thumb domains. The 5′-end RNA
cytosine (rC1) and uracil (rU2) are located at the interface of the
fingers and palm domains and are involved in a network of inter­
actions with conserved residues (Supplementary Fig. 4) of motifs 2
and B′, both of which are located in proximity to the active site of
the enzyme (Fig. 2a,b). In particular, the 2′-OH of rC1 is within
hydrogen bonding distance of the backbone carbonyls of Val197
of motif 2 and Gly309 of motif B′, and the pyrimidine base sits
over the otherwise solvent-exposed hydrophobic side chain of the
­conserved Ile196, which also forms part of motif 2 (Fig. 2b). Contacts
between rU2 and the protein are mediated by the short aliphatic
side chain of Pro311 and the ribose group (Fig. 2b). Interactions
between rC1 and rU2 with motifs 2 and B′ place the cytosine base in
proximity to the active site of the enzyme, where it is well positioned
for Watson-Crick base pairing with the incoming nucleotide substrate. Stabilization and placement of the 5′-end bases of the templating region above the active site of the enzyme are further facilitated by
the interactions of the remaining five ribonucleotides with the incoming DNA primer. Limited contacts between this part of the RNA and
the protein are mediated via a water molecule, which coordinates the
2′-OH of guanosine (rG3) with the backbone of helix α15 (Fig. 2b).
Notably, the structural organization of helix α15 is influenced by the
IFD motif, a long insertion consisting of two helices (α13 and α14),
between motifs A and B′ (Fig. 2a), which explains why mutations in
this motif lead to loss of telomerase function1,37.
Contacts between TERT and the DNA substrate are mostly
mediated via backbone interactions with the thumb loop and
helix (Fig. 3a). The thumb helix1 sits in the minor groove of the
RNA-DNA heteroduplex, making extensive contacts with the
phospho­diester backbone and the ribose groups of the RNA-DNA
hybrid. The mode of action of the thumb helix of telomerase appears
to be similar to that proposed for the equivalent helix (helix H) in
retroviral reverse transcriptases38,39. Another conserved element of
the thumb domain, known as the thumb loop1, runs almost parallel
to the curvature of the DNA primer, and the two are involved in a
network of backbone- and solvent-mediated interactions (Fig. 3b).
Interactions between the DNA and the thumb loop include the
side chains of Lys416 and Asn423, both of which extend toward the
center of the ring and are within hydrogen bonding distance of the
DNA backbone. Contacts between the thumb domain and the DNA
position the nucleotides located at its 3′ end within co­ordinating
distance of the primer-grip region, a short, rigid loop located at
the interface of the palm and thumb domains and in proximity
to the active site of the enzyme (Fig. 4a,b). The tip of this loop,
formed by the backbone of conserved residues Cys390 and Gly391
(Supplementary Fig. 4b), abuts the ribose group of C22. This
interaction guides the 3′-end DNA nucleotides toward the active
site of the enzyme (Fig. 4b). The active site of the enzyme, where
the incoming nucleotide is projected to bind1, is partially occupied
by the nucleotide (G24) located at the 3′ end of the DNA (Fig. 4b).
The ribose group and, to a certain extent, the guanosine base of G24,
which makes Watson-Crick pairing interactions with the rC1 located
a
b
C17
A18
RNA
DNA
Wat6
G19
Thumb
loop
Thumb
helix
DNA Asn423
G20
Thumb
loop
T21
Wat9
Lys416
Figure 3 TERT–telomeric DNA associations. (a) TERT-DNA (yellow stick)
binding is mediated by the thumb loop (light blue) and helix (light blue).
The thumb helix docks itself into the minor groove of the RNA-DNA
hybrid, and the thumb loop adopts the geometry of double-stranded
nucleic acid and is involved in a network of interactions with the DNA.
(b) Interactions between the thumb loop and the DNA are mostly mediated
by backbone and solvent. Also, the side chains of Lys416 and Asn423
(light blue stick) that form part of the thumb loop extend toward the
center of the ring and coordinate the DNA backbone.
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a
b
© 2010 Nature America, Inc. All rights reserved.
Figure 4 DNA interactions with the primer
DNA
DNA
DNA
grip region and the active site. (a) Overview of
T21
T21
Thumb
the interactions of the thumb domain (thumb
Primer Gly391
Primer Gly391
loop
grip
RNA
grip
C22
C22
helix and loop; light blue), the primer-grip
A23
A23
region (magenta loop) and the active site of
G24
G24
TERT (three aspartates; stick) with the DNA
Val342
Val342
Cys390
Cys390
substrate (yellow stick). The view is a 90°
3′
Asp344
Asp344
rotation of Figure 3a. (b) Stereo view of the
Active
Asp251
Asp251
DNA interactions with motif E and the activeAsp343 3′
site
Asp343 3′
Gln308
Gln308
site residues. The tip of the primer-grip region
Active
Active
Thumb
Tyr256
Tyr256
Primer
grip
site
Ile252
site
Ile252
helix
(magenta loop), formed by the backbone of
(motif E)
residues Cys390 and Gly391, abuts the ribose
group of C22; this interaction guides the 3′ end of the DNA at the active site of the enzyme for nucleotide addition. The active site–bound magnesium
ion (dark red sphere) coordinates the DNA backbone formed by the last two nucleotides. The nucleotide binding pocket of TERT, which is partially
occupied by the last DNA nucleotide, is in part formed by the highly conserved residue Val342 and the invariant Tyr256 and Gly308.
at the 5′ end of the RNA template, sit in a well-defined hydrophobic
pocket formed by the side chains of the invariant Tyr256 and Gln308
and the conserved Val342 (Supplementary Fig. 4a,b) of motifs A, B′
and C, respectively; the α-phosphate is coordinated by the Mg2+ ion
occupying the active-site aspartates (Fig. 4b). The important role of
Val342 in telomerase selectivity has been previously shown for the
human telomerase holoenzyme40.
TERT domain rearrangements upon nucleic acid binding
Comparisons of the nucleic acid–bound and substrate-free TERT
structures suggest that TERT–nucleic acid associations induce small
rigid-body changes in orientation between subunits of the enzyme
that lead to a 3.5-Å decrease in the diameter of the interior cavity of
the ring. The decrease arises from a 6° inward rotation together with a
3.5-Å translation of the thumb domain with respect to the fingers and
palm domains (Fig. 5a). Translation of the thumb domain toward the
center of the ring is accompanied by the TRBD, which is also shifted
3.5 Å toward the finger domain, creating a more narrow RNA binding
pocket than that of the substrate free-enzyme (Fig. 5b). The precise
role of this subtle structural rearrangement is unclear at this point;
however, it may have significant implications for TERT association
with the full-length RNA, TER.
DISCUSSION
TERT contains discrete domains that carry out the mechanically
complicated reaction of nucleic acid and nucleotide binding and
selectivity in a coordinated manner during telomere replication. The
structure presented here provides a detailed picture of the physical
contacts between TERT and nucleic acid substrates, which in turn
provides mechanistic insights into RNA-template and DNA binding
required for telomerase-elongation complex formation.
Common aspects of substrate binding between TERT and HIV RTs
It has been postulated that telomerase uses a mechanism of DNA replication that resembles that of other retroviral reverse transcriptases,
a theory supported by the structure of the TERT–nucleic acid complex presented here. Structural comparison of the RNA-DNA–bound
TERT and HIV RT36 (PDB 1HYS) shows a striking similarity in the
overall domain organization and nucleic acid binding between the
two structures (Fig. 6a). As with HIV RTs, telomerase-dependent telo­
mere replication requires the pairing of the templating region with
the incoming DNA primer and the placement of the 3′ end of the
DNA into the enzyme’s active site for nucleotide addition. Moreover,
TERT– or HIV RT–nucleic acid associations are accompanied by
domain rearrangements that facilitate the formation of a tight, catalytic, protein–nucleic acid assembly and the positioning of the DNA
516
3′ end at the active site of the enzyme for catalysis39,41,42. Contacts
between TERT or HIV RT and the RNA-templating region are specific
and involve key signature motifs (motif 2 and B′ of the fingers and
palm domains, respectively) that are ­hallmarks of these families of
enzymes, and these motifs are required for positioning of the solventaccessible bases of the RNA template in proximity to the active site for
nucleotide binding and selectivity. Contacts between TERT or HIV RT
and the DNA substrate are mediated in part by the thumb domain,
and despite the lack of sequence homology in this region between the
two families of enzymes, the mode of action of the thumb helix of
telomerase is similar to that proposed for helix H of HIV RTs38,39,43
(Fig. 3a). Placement of the DNA 3′ end at the active site of the enzyme
is further facilitated by the primer-grip region, another highly
­conserved motif between TERT and HIV RTs44 (Fig. 4a,b).
Although we were unable to trap the enzyme in its catalytic state,
the partially occupied (by the nucleotide G24) active site of the
enzyme, formed by a number of invariant (Asp251, Tyr256, Gln308,
Asp343, Asp344) or highly conserved (Val342) residues located at the
3′ end of the DNA (Fig. 4b), gives us a glimpse into the mechanism of
­nucleotide binding and selectivity of telomerase during the replication
process. Strikingly, the invariant Tyr256 and Gln308 are also present
in HIV RTs (Fig. 6b), where, as in TERT, they are involved in nucleotide binding and selectivity through positioning for interactions with
the templating region35,45, further supporting common mechanistic
aspects of DNA replication between these families of enzymes.
TERT rigid conformational changes and function
Domain reorganization upon nucleic acid binding is a common
feature of RNA and DNA polymerases as well as retroviral reverse
­transcriptases, and this process is geared toward both the formation
a
Thumb
b
Palm
3.5 Å
Palm
6°
90°
Fingers
TRBD
Fingers
Figure 5 TERT rigid conformational changes upon nucleic acid binding.
(a) Structural comparisons of the substrate-free1 (PDB 3DU6; green
cartoon) and substrate-bound TERT molecules (color scheme as in
Fig. 1d) show that nucleic acid binding in the interior cavity of the
TERT ring is accompanied by a 6° upward rotation and a 3.5-Å inward
translation of the thumb domain. For clarity, the TRBD domain has been
removed from this figure. (b) Overlay of the two structures also shows that
in the nucleic acid–bound TERT structure the TRBD has moved closer to
the finger domain by 3.5 Å.
VOLUME 17 NUMBER 4 APRIL 2010 nature structural & molecular biology
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© 2010 Nature America, Inc. All rights reserved.
a
b
the RNA does not directly engage the RNA
binding pocket of TRBD. The structure shows
Val342/Met184
that TERT-RNA contacts position the 5′ end
Gly391/Gly231
of the templating region at the entry of the
Motif
Tyr256/Tyr115
B′
Motif
RNA binding pocket of TRBD (Fig. 2a).
Primer
C
grip
This
arrangement would place either the
Gly309/Gly152
template boundary element51–53, present in
Thumb
most organisms, or the short oligonucleotide
Active
Gln308/Gln151
Motif
overhang of rodent TER51 within the RNA
site
HIV RT
A
Fingers
binding pocket of TRBD. The stable associaTERT
tion of TER with the TRBD would force the
Figure 6 Structural comparison of the RNA-DNA–bound TERT and HIV RT. (a) Structural
enzyme
to stall when reaching the nucleotide
comparison of TERT (yellow) bound to RNA-DNA (orange) and HIV RT (light blue) bound to
located at the 5′ end of the RNA template,
RNA-DNA (blue) (PDB 1HYS)36. The TRBD has been removed for clarity. (b) Overlay of the palm
thus preventing replication beyond this point.
domains of the RNA-DNA–bound structures36 of TERT and HIV RT (PDB 1HYS) shows a strong
conservation in the sequence and organization of the primer grip and active-site residues.
Stalling of the enzyme for extended periods
Active-site residues of TERT (left of slash) and HIV RT (right of slash) are labeled.
would lead to destabilization and dissociation
of the RNA-DNA heteroduplex and initiation
of another round of telomere replication.
of a tight, catalytic protein–nucleic acid assembly and the positioning
Collectively, the data presented here, together with previous strucof the DNA 3′ end at the active site of the enzyme for catalysis39,41,42. tural and biochemical evidence, support common mechanistic aspects
Unlike HIV RTs, telomerase seems to exist, at least in the absence of the of substrate binding and DNA replication between telomerase and
full-length integral RNA component, in a closed-ring configuration, HIV RTs, suggesting an evolutionary link between these families of
an arrangement mediated by extensive contacts between the TRBD enzymes. These data also provide novel insights into the basic mechaand the thumb domains1. Comparison of the nucleic acid–bound nisms of telomere replication and length homeostasis by ­telomerase,
and substrate-free TERT structures suggests that TERT–nucleic acid which in turn enriches our understanding of the mechanism of
associations induce subtle rigid-body changes in orientation between DNA replication by polymerases in general. Moreover, the structure
subunits of the enzyme, leading to a 3.5-Å decrease in the diameter presented here provides a detailed picture of the physical contacts
of the interior cavity of the ring.
between TERT and its nucleic acid substrates, information that is
These observations are unexpected because, in most polymerases, invaluable in our effort to design small-molecule inhibitors of telo­
including the HIV RT, the fingers and thumb domains undergo sig- merase that may be of therapeutic value for cancer and other diseases
nificant conformational changes required for substrate binding and associated with cellular aging.
function42,46,47. For example, the fingers domain, which is known
to bind and position the nucleotide at the active site of the enzyme, Methods
undergoes significant conformational changes referred to as the Methods and any associated references are available in the online
‘open’ and ‘closed’ states46. It is therefore possible that the interactions version of the paper at http://www.nature.com/nsmb/.
between the TRBD and the thumb domain lock the fingers domain in
place, thus preventing the conformational rearrangements observed in Accession codes. Protein Data Bank: Coordinates for the TERT–RNAother polymerases, which would suggest the possibility of a preformed DNA complex have been deposited under accession code 3KYL.
active site. A preformed active site has been previously observed for
the hepatitis C viral RNA polymerase48, a close structural homolog of Note: Supplementary information is available on the Nature Structural & Molecular
Biology website.
TERT, and also for the Y-family DNA polymerases49. Another possibility is that we trapped the substrate-free TERT enzyme in the closed Acknowledgments
fingers conformation. Assuming this is true, significant movement We would like to thank S.J. Gamblin and S. Pennell for helpful discussions.
of the fingers domain of TERT would most likely require the TRBD Funding for this project was generously provided by the Ellison Medical and the
V Foundations as well as the Pennsylvania Department of Health.
and the thumb domain to be splayed apart. Contacts between the
TRBD and the thumb domain are extensive—thus, significant energy AUTHOR CONTRIBUTIONS
would be required to force them apart1. This could be accomplished E.S. designed the experiment plan, analyzed the data and wrote the manuscript;
by accessory proteins that possibly act in a similar manner to that of A.G. prepared the recombinant telomerase and carried out the reverse transcriptase
assays; M.M. carried out the TRAP assays; H.F. and M.F. provided advice with the
the sliding clamp loader of DNA polymerases50.
T. castaneum TRAP assays.
RNA
DNA
Palm
Cys390/Met230
Repeat addition processivity
Telomerase, unlike most polymerases, has the ability to add multiple
identical repeats of DNA to the ends of chromosomes, a process
known as repeat addition processivity. This unique characteristic
of telomerase has been attributed in part to the association of the
N-terminal portion of TERT with TER and the telomeric overhang as
well as the IFD motif 37. The TEN domain, present in several organisms, and its weak interaction with the DNA substrate is thought to
be a determinant of repeat addition processivity15–17; most recently,
the TRBD and its stable association with TER has also been shown
to be involved in this process11,12. In the complex presented here,
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The authors declare no competing financial interests.
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VOLUME 17 NUMBER 4 APRIL 2010 nature structural & molecular biology
ONLINE METHODS
Protein expression and purification. We overexpressed the wild-type and mutant
(D251A) T. castaneum, full-length TERT proteins with some subtle modifications
to the protocol used previously1 that proved useful in increasing the protein
yield. We now overexpressed the protein in Escherichia coli Rosetta (DE3) pLysS
(Novagen) at 30 °C for 5 h. We dialyzed stock protein (10 mg ml−1) in 10 mM
Tris-HCl, 100 mM KCl, 1 mM TCEP, pH 7.5, before crystallization trials.
© 2010 Nature America, Inc. All rights reserved.
Preparation of T. castaneum extracts and total RNA isolation. We ground 20
T. castaneum larvae or pupae in liquid N2, homogenized them with 200 µl extraction buffer (25 mM Tris-HCl, 5 mM β-mercaptoethanol (β-ME), 1 mM EGTA,
0.1 mM benzamidine, 200 mM KCl, 10% (w/v) glycerol, 10 mM imidazole and
RNasin (Promega), pH 7.5) and placed them on ice for 30 min. After we centrifuged the homogenate at 12,000g at 4 °C for 20 min, we collected the supernatant, flash-froze it in liquid nitrogen and stored it at −80 °C before use. We then
extracted the total RNA from the T. castaneum homogenate using the RNeasy
Protect Mini Kit from Qiagen.
In vitro reconstitution of T. castaneum telomerase. The telomerase RNA of
T. castaneum is unknown, so we used the total RNA isolated from T. castaneum
larvae and the recombinant TERT to assemble the telomerase complex in vitro.
We mixed 20 µg of the His-tagged TERT (25 mM Tris, 200 mM KCl, 10% (w/v)
glycerol, 5 mM β-ME and 10 mM imidazole, pH 7.5) with 50 µl of T. castaneum
larvae total RNA, and we incubated the two in T. castaneum lysate for 2 h at
22 °C in the presence of RNasin. We then purified the telomerase ­complex over
a nickel–nitrilotriacetic acid column and tested it for activity using a ­modified
version of the TRAP assay54 as described below.
Telomerase repeat amplification protocol assays. We tested the activity of the
in vitro–reconstituted T. castaneum telomerase using the following TRAP assay. We
carried out the telomerase-elongation step in a 50-µl reaction mixture composed
of 20 mM Tris-HCl (pH 8.3), 7.5 mM MgCl2, 63 mM KCl, 0.05% (w/v) Tween
20, 1 mM EGTA, 0.01% (w/v) BSA, 0.5 mM of each dNTP, 1 µM DNA primer
(5′-AAGCCGTCGAGCAGAGTC-3′) (Tcas-TS)) and 4 µg of nickel–­nitrilotriacetic
acid–purified T. castaneum telomerase. After incubation at 30 °C for 60 min,
we extracted each reaction mixture with phenol-chloroform and precipitated
each with ethanol. We resuspended each sample in 50 µl PCR reaction buffer
(10 mM Tris-HCl (pH 8.0), 50 mM KCl, 2 mM MgCl2, 100 µM dNTPs (dATP,
dTTP and dGTP), 10 µM [32P]dCTP (80 Ci mmol−1), 1 µM Tcas-CX primer
(5′-GTGTGACCTGACCTGACC-3′) and HotStar Taq DNA polymerase (Qiagen)).
We resolved the PCR products on Tris-borate-EDTA (TBE)–polyacrylamide gels
(Supplementary Fig. 1). We also confirmed the presence of multiple telomeric
repeats (TCAGG)n by subcloning and sequencing the TRAP products.
TERT, reverse transcriptase assays. We carried out standard reverse transcriptase
assays using the recombinant T. castaneum TERT and the RNA-DNA hairpin
used in this study to test TERT’s ability to replicate the end of the DNA substrate
that comprises part of the RNA-DNA hairpin. We carried reverse transcriptase
assays out in telomerase buffer (50 mM Tris-HCl, 100 mM KCl, 1.25 mM MgCl2,
doi:10.1038/nsmb.1777
5 mM DTT, 5% (w/v) glycerol, pH 8 at room temperature), 100 µM dNTPs
(dATP, dTTP and dGTP), 10 µM [32P]dCTP (80 Ci mmol−1), 5 µM RNA-DNA
hairpin and 1 µM recombinant TERT. We incubated the samples for 2 h at room
temperature and then extracted and precipitated them with phenol-chloroform
and ethanol, respectively. We resuspended the DNA pellet in a solution composed of 90% (w/v) formamide and 10% (w/v) glycerol and ran the sample on
a 12% (w/v) polyacrylamide–7 M urea gel in 1× TBE at 220 V for 70 min at 4 °C
(Supplementary Fig. 3).
Protein crystallization and data collection. We prepared the binary complex
by adding to the dialyzed protein 1.2 M excess nucleic acid (RNA-DNA hairpin purchased from Integrated DNA Technologies), 5 mM dNTPαS (Jena
Biosciences GmbH) and 5 mM MgCl2. Crystals of the monoclinic space group
P21 that diffracted to 2.7-Å resolution appeared in 3 d and grew to final size
in 2 weeks. We grew crystals by the vapor-diffusion, sitting-drop method by
mixing one volume of the ternary complex with one volume of reservoir solution containing 0.1 M HEPES (pH 7.5) and 12% 1,6-hexanediol or PEG 4K and
0.2 M KCl. We transferred crystals into cryoprotectant solution that contained
0.1 M HEPES (pH 7.5), 15% (w/v) 1,6-hexanediol or PEG 4K, 15% (w/v)
glycerol, 0.2 M KCl and 1 mM TCEP and harvested them by flash freezing in
liquid nitrogen. We collected X-ray data at the NSLS beamline X25 and processed
it with MOSFILM as implemented in WEDGER-ELVES55 (Table 1). There is one
monomer in the asymmetric unit.
Structure determination and refinement. We calculated phases by molecular
replacement with PHASER56, using the substrate-free TERT structure1 (PDB
3DU6) as a search model. Maps calculated after one cycle of refinement by
CNS-SOLVE57 revealed clear 2Fo − Fc density for all 596 residues of TERT and
Fo − Fc density for the nucleic acid substrate at 3.0s contour level. We carried out
model building in Coot58 and refined the model using both CNS-SOLVE and
REFMAC559. We carried out the last cycles of refinement with TLS restraints as
implemented in REFMAC5. We refined the structure to good stereochemistry,
with 84.3, 14.3 and 1.4 of the residues in the most favorable, additional allowed
and generously allowed of the Ramachandran plot, respectively.
54.Sasaki, T. & Fujiwara, H. Detection and distribution patterns of telomerase activity
in insects. Eur. J. Biochem. 267, 3025–3031 (2000).
55.Holton, J. & Alber, T. Automated protein crystal structure determination using
ELVES. Proc. Natl. Acad. Sci. USA 101, 1537–1542 (2004).
56.Potterton, E., Briggs, P., Turkenburg, M. & Dodson, E. A graphical user interface
to the CCP4 program suite. Acta Crystallogr. D Biol. Crystallogr. 59, 1131–1137
(2003).
57.Brunger, A.T. et al. Crystallography & NMR system: A new software suite for
macromolecular structure determination. Acta Crystallogr. D Biol. Crystallogr. 54,
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