Subunit TERT in Tribolium castaneum
'15 and Nicole Valentini '14
The telomerase catalytic subunit
in flour beetle, Tribolium castaneum
is a specialized ribonucleoprotein
(RNP) reverse transcriptase
extending the 3' ends of eukaryotic linear chromosomes, which helps
genomic stability and cell viability. It binds to RNA templates and
telomeric DNA. Lack of telomerase could lead to a reduction in cell size
and cellular aging.
Telomerase is capable of adding multiple identical repeats of DNA.
Initiation of telomere replication begins with the pairing of an
RNA-templating region with an incoming single-stranded DNA primer.
Completion of the telomere extension leads to the dissociation of the
RNA-DNA hybrid. The DNA is then repositioned so that the DNA end is at
the active site of TERT. The RNA-DNA pairing is at the other end of the
template. Regulated by telomere binding proteins, telomerase is
associated with the chromosome end until several telomeric repeats have
II. General Structure
TERT is structurally similar to several other eukaryotic and
prokaryotic binding proteins, namely RNA polymerases and B-family DNA
polymerases. Like these proteins, it mimics the structure of a hand
with finger, palm, and thumb domains essential for nucleotide
associations and catalysis. The finger
domain is involved in nucleotide binding and
, the palm
domain contains the active site of the
, and the thumb
domain is involved in DNA binding and
. These domains are organized into a ring configuration as seen in a
typical substrate free enzyme. The image presented represents a
crystallized full length active T.
castaneum TERT with an RNA-DNA hairpin containing both an RNA-templating
and a complementary
, joined together by an RNA-DNA linker.
TERT binding to the RNA-template is mediated by
2 and B'
of the palm domain, which localize
adjacent to and above the active site of the enzyme.
Protein contacts with the backbone of the RNA template position the
bases within the active site of the enzyme for nucleotide binding,
allowing for selectivity. In general, TERT DNA interactions are
mediated by the thumb loop and helix, which provide the stability for
functional telomerase elongation and facilitate the positioning of the
3'-end nucleotides near the primer-grip region (also called motif E).
Furthermore, motif E positions the 3'-end hydroxyl of the DNA primer
at the active site
of the enzyme, facilitating nucleotide addition.
Overall, interactions between TERT, the RNA-templating region, and the
DNA primer extended in elongation are important for providing more
insight into how telomeres are replicated.
The RNA component of T. castaneum was designed from previous
information, as its exact composition is not known. The RNA-DNA
hairpin contains three-nucleotide overhangs at the 5' end of the RNA
template, in order to keep the enzyme in its catalytic state when
and non-hydrolyzable nucleotides were used to facilitate
co-crystallization of the protein-nucleic acid assembly.
III. RNA Binding
The DNA substrate
with TERT is usually associated with the RNA-templating region TERT,
which is an active polymerase in the presence of nucleic acids. The
RNA template makes contact with conserved motifs. TERT also makes
contact with the RNA template, which positions the solvent-accessible
bases adjacent to the active site of the enzyme, allowing nucleotide
binding. The TERT-RNA association further positions the 5' end of the
RNA template at the RNA binding pocket. This is where the template
boundary element, which is upstream of the RNA-template, binds. The 3'
end of the DNA substrate at the active site of the enzyme is placed at
the 3' end of the DNA substrate, allowing nucleotide addition to be
Interactions with the RNA-templating region are facilitated by the
fingers, palm, and thumb domains. The 5'-end of RNA is
in close proximity to the active site of the enzyme. Specifically, the
2'-OH cytosine is close enough to engage in hydrogen
bonding with the backbone carbonyls of
of motif 2 and Gly309
of motif B', placing cytosine near the active site. The incoming
nucleotide substrate is then ready to base pair with it. Contact with
uracil and the protein are aided by the short aliphatic side chain of Pro311
and the ribose group.
The 5'-end bases of the templating region are stabilized with five
ribonucleotides that are attached to the incoming DNA primer. Water
molecules limit the contact between this
region and TERT.
IV. DNA Binding
between TERT and the DNA substrate are mediated with backbone
interactions and the thumb loop and helix. The thumb helix makes
contact with the phosphodiester backbone and ribose groups of
the RNA-DNA hybrid, sitting in the minor groove. The thumb loop,
which is a part of the thumb domain,
sits parallel to the twist of the DNA primer, creating a network
These interactions include the side chains of Lys416 and
Asn432, both of which can hydrogen
bind with the DNA backbone.
The thumb domain's contact with DNA positions the nucleotides at
the 3' end within the primer-grip region, which is formed by the
backbone of residues Cys390
. This guides the 3'-end DNA nucleotides towards the active site
of the enzyme. The TERT active site consists of three amino acid
which form parts of two short loops that are located on the palm
Residues Tyr256 and
form hydrophobic pockets that are adjacent to three catalytic
apartates, and accommodate the base of the nucleotide substrate.
The TRBD domain forms a deep cavity on the surface of TERT that forms
a gap allowing for nucleotide entry into the hole of the ring.
This hole is important as it forms during TERT-TER assembly and
it serves as the area for the placement of the RNA template in
the interior of the ring.
When TERT is bound to nucleic acids, there is a small
conformational change in the interior cavity of the ring. The
diameter's size decreases, which could be related to TERT's
association with TER (full-length RNA). More specifically, a
change in the thumb domain and the TRBD toward the center of the
ring creates a more narrow binding pocket than that of a
substrate free TERT. Though not very clear, it is believed that
this structural change is essential for the association of TERT
V. Telomere Replication
Repeat addition processivity
is a process where telomerase adds multiple identical repeats of
DNA to the ends of chromosome. This process is highly
facilitated by the enzyme's integral RNA-templating
region (TER). When telomere replication is initiated,
pairing of the RNA-templating region with the incoming single
stranded DNA primer occurs. Evidence suggests that the RNA
template serves as the facilitator for several rounds of
nucleotide addition and selectivity.
Telomerase is able to add multiple identical repeats to
telomeres mainly by the association of the N-terminal
portion of TERN with TER,
the telomeric overhang, and the IFD motif.
domain (which is not shown, but is indicated in pink in
the link) is believed to be an important factor in addition
processivity, as well as the association of TRBD with TER.
Associations between TER and TRBD force the enzyme to stall when
reaching the nucleotide located at the 5' end of this RNA
template, halting replication. If this stalling occurs for long
periods of time, the complex is destabilized and dissociation of
the RNA-DNA heteroduplex results in the initiation of another
round of telomere replication.
VI. TERT and HIV RTs
TERT-nucleic acid associations and domain organizations are similar
to the structures observed for HIV RTs. In both systems,
pairing of the templating
with the incoming DNA primer is necessary. Furthermore, the 3'-end of
the DNA needs to be placed in the enzyme's active site in order for
nucleotide addition to occur. Higher affinity binding also occurs in
both structures due to domain rearrangements - the 3'
end of DNA
is more tightly bound to the active site during
the enzymes' catalysis.
Both TERT and HIV RT involve the 2 and B' of the fingers and palm
domains motif. These positions assist the positioning of the
solvent-accessible bases of the RNA template so that it is close to
the active site for nucleotide binding. When either TERT or HIV RT
interacts with DNA,
the substrate is mediated by the thumb domain.
Common mechanistic aspects of DNA replication, such as these, are seen
in both substrates.
Gillis, Andrew J.,
Anthony P. Schuller, and Emmanuel Skordalakes. 2008. Structure
of the Tribolium castaneumi telomerase catalytic
subunit TERT. Nature Structural and Molecular Biology
Meghan, Andrew Gillis, Mizuko Futahashi, Haruhiko Fujiwara,
and Emmanuel Skordalakes. 2010. Structural basis for
telomerase catalytic subunit TERT binding to RNA template and
telomeric DNA. Nature Structural and Molecular Biology
Osanai, Mizuko, Kenji K. Kojima, Ryo
Futahashi, Satoshi Yaguchi, and Haruhiko Fujiwara. 2006.
Identification and characterization of the telomerase
reverse transcriptase of Bombyx mori (silkworm) and
Tribolium castaneum (flour beetle). Gene Section
Evolutionary Genomics 376: 281-289.
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