Overview

1. Telomere and End Replication Problem

Structure and Function of
Telomerase 2. Function of Telomerase

3. General structure of Telomerase

By Peng Wang & Yuzhen Wang
May 11,2006 4. Telomerase Essential N-terminal (TEN)
Domain

5. Regulation of Telomerase Activity

Telomere Function
Telomeres
Telomeres function by protecting chromosome ends
from recombination, fusion to other chromosomes, or
• Telomeres are the structure at the end of degradation by nuclease.
linear eukaryotic chromosome.
Allow cell to differentiate between natural
chromosome ends and damaged DNA.

Provide Mechanism for Replication of Linear DNA

Ends

Molecular Biology of the Cell, 4th edition

Telomere Function
Structure of Telomeres
Telomeres function by protecting chromosome ends
from recombination, fusion to other chromosomes, or
degradation by nuclease. Telomere are composed of tandem repeats of GC-
rich DNA sequence and telomere associated protein.
Allow cell to differentiate between natural In human, the repeat is TTAGGG, up to 10,000bp in
chromosome ends and damaged DNA. length.

Provide Mechanism for Replication of Linear DNA Telomeres have a 3’ G-rich overhang
Ends

1
 t-loop of telomere Replication Problem of End of Linear Chromosomes

Molecular Biology of the Cell, 4th edition

Cell 1997, 97:419-422 Molecular Biology of the Cell, 4th edition

http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/T/Telomeres.html#Replication_of_linear_chromosomes
_presents_a_special_problem.

Telomerase

Telomerase is a
specialized
reverse
transcriptase,
contains both
RNA and
Protein

Science 1997, 276: 561-567

Annu.Rev.Biochem. 2006, 75:493-517

2
 Unique features of telomerase Structure and Evolution
of TERT

Two components---- telomerase reverse

transcriptase(TERT) protein and telomerase
RNA
Internal RNA serves as template for reverse
transcription by TERT
Ability to translocate along the DNA

Annu.Rev.Biochem. 2006, 75:493-517

RT domain of TERT C-terminal domain of TERT

Mediate the addition of nucleotide.

Consistute the “thumb” domain of telomerase,
Three Asp residues in RT motifs A and C of Weak consevation of sequences
TERT are important for catalysis of nucleotide
Multiple mutations in this domain of both
addition, two-metal catalysis mechanism. yeast and human
Might be involved in nucleotide addition and
Sizable insertion between motif A and B’(IFD) processivity
invovle in telomerase activity.

Science 1997, 276:561-567

Telomerase RNA Crystal Structure of the Essential N-terminal

The template region of the telomerase reverse transcriptase (TEN)
Nature structureal & molecular biology 2006, 13: 218-225
Main TERT-binding
region
1. Recombinant TEN (2-191 A.A.) from
Template boundary Tetrahymena , with a HIS6 tag, was purified from
element(TBE) E.coli and crystalized with 3 molecules in
crystallographic asymmetric unit.
TRE, Template 2. Telomerase activity assay, DNA binding assay,
recognition element
RNA binding assay to compare the wild type,
Low affinity TERT
TEN deleted or TEN mutant TERT
binding sites 3. A conceptual model

Annu.Rev.Biochem
2006, 75:493-517

3
 Telomerase Essential N-terminal (TEN)
Domain of TERT from Tetrahymena Conservation of TEN domain structure

(a) Multiple sequence alignment

of the TEN domains from
selected TERT homologs. red
(a) Location of TEN within TERT residues, 100% conservation;
green, >60% conservation; blue,
(b)Stereo ribbon diagram of the TEN domain: similarity but not identity.
a core βsheet, 4 anti-parallel βstrands, a <20% identity, but significant
βhairpin and 7 αhelix, 2 long loops. similarity.
(c) Topology diagram of the TEN domain. Gly144: connects α5 and α6
GLy171: β6 and α7
H-bonds betweenβ 2 and β5, α3 and α6, α2 Gln168: on surface ---catalytic
and α5 Activity.
(b) Mapping of conserved
(d) Superposition of backbone atoms of the
residues onto the surface of the
three TEN molecules found in the TEN domain.
crystallographic asymmetric unit. Dashed Some conserved residues make a
lines, disordered portions of the C termini of groove form C tail to center. And
chains B and C. Flexible? Need partener? others have hydrophobic
character.

Nature structureal & molecular biology 2006, 13: 218-225

Recombinant TEN (2-191 A.A.), with a HIS6 tag, was purified from E.coli and
crystalized with 3 molecules in crystallographic asymmetric unit.

Interaction of TEN with telomeric iodinated DNA—photo-cross-linking

In vitro telomerase activity of TEN
mutants
(a) Direct primer-extension
assay of telomerase activity
for TEN domain mutants.
-TEN
Q168: groove
F178, W187: c-tail?

(b) Quantification of
telomerase activity for each
mutant.

IU: 5-iodouridine substitutions far from the 3’ end

(a) Specific binding between purified TEN domain protein and telomeric DNA: specific to telomeric DNA
(b) w/wo RNA subunit of TERT or TERT with the TEN deletion (equal mixture): TEN and TR increase the binding
(c) Site-specific mutant of TERT: TEN increase binding , mutation decrease binding
(d) Wild type/ mutant TERT mixed with TEN deleted TERT: Q168, F178, W187
(e) Quantification of the amount of telomeric DNA cross-linked to each mutant TEN domain
(f) Surface representation of TEN domain with the three residues essential for telomeric DNA cross-linking in red

Conceptual model of contributions of the

TEN domain to the telomerase mechanism

TEN domain (yellow) binds a

portion of the telomeric DNA primer
(thick arrow) that is distant from
the 3' end (arrowhead), which
undergoes extension in the
reverse-transcriptase domain of
TERT (red).

The TEN domain also interacts with

the RNA subunit TR; these
interactions are crucial for catalysis
but contribute little to binding,
which is accomplished mainly by
the RNA-binding domain of TERT
RNA-binding activity of the TEN domain (green).
(a) RNA binding of TERT2-191 and TERT13-184: non-specific RNA binding but require full length 2-191
(b) Electrostatic surface potential of the TEN domain , positively charged C-terminal
(c) Binding of TR to the TEN domain protects the N and C termini for limited proteolysis by Lys-C.
The Ends of TEN are disordered in solution and subject to Lys-C digestion.

4

Regulation of Telomerase Activity
1. Transcriptional level
hTERT instead of TR is the rate-limiting factor for assembly of an active
telomerase complex
Myc/Mad, Estrogen and androgen upregulate TERT expression
Tumor suppressor gene(WT1) downregulate TERT expression
2. Postranslation
• Folding: p23, Hsp90
• Dimerization: believed to be more active
• Modification
Phosphorylation: by PKC, AKt, upregulate activity
Ubiquitination: by MKN1, a ubiquitin ligase, degradation
• Translocation : cytoplasm to nucleus: A.A. 1-15, 326-620
• Direct protein binding: Ku↑, hEST1↑, Kip↑and PinX1↓
• Telomere length: Shortening of telomere activate telomerase

Telomerase activity

Hsp90
Adrogen
PinX1 PKC AKt
MKN1 WT1
Estrogen

Aging Cancer