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Telomerase RNA

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Telomerase RNA (TER): Sequential and Structural Characteristics ... Tools and Specific Problems of Yeast TER ... The Problems in the case of yeast TER: ... – PowerPoint PPT presentation

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Title: Telomerase RNA


1
Telomerase RNA
from sequence to structure
Elhanan Elboher Dr. Dudy Tzfati, Dept. of
Genetics, HUJI
2
The Telomerase RNP complex adds short DNA repeats
to the end of the chromosome
3
The Telomerase RNP complex adds short DNA repeats
to the end of the chromosome
4
The Telomerase RNP complex adds short DNA repeats
to the end of the chromosome
5
The Telomerase RNP complex adds short DNA repeats
to the end of the chromosome
6
The Telomerase RNP complex adds short DNA repeats
to the end of the chromosome
7
Telomerase RNA (TER) - Functions
  • Contains the template for the telomeric repeats
  • Regulates the length of the transcribed sequence
    (by a template boundary element)
  • Scaffold that the elements of the complex (TERT
    and other factors) are bound to
  • Regulates the activity of the whole complex, by a
    switch between different states

8
Telomerase RNA (TER) Sequential and
Structural Characteristics
  • Divergent length (Vertebrates 450 nt, Ciliates
    150 nt, Yeast 1000 nt)
  • Low conservation of the sequence, even among
    closed species
  • Higher conservation of short subsequences which
    are involved in formation of functional
    structural elements
  • General structure in Yeast species three long
    arms and a central catalytic core, contains an
    essential pseudoknot element

9
Saccharomyces
Secondary Structure Models of Two
Yeast TER Groups
Kluyveromyces
10
Conserved Sequences in the Kluyveromyces Group
11
My Project - Objectives
  • Focus on a third yeasts group, Candida
  • Finding the exact start end points of the
    folded sequence
  • Structural elements which are known as essential
    from other species (template boundary, protein
    binding domains, ?-knot etc.)
  • Novel structural elements
  • Contribute to the general model of the secondary
    and tertiary structure of the yeast telomerase RNA

12
My Project - Objectives
  • Focus on a third yeasts group, Candida
  • Finding the exact start end points of the
    folded sequence
  • Structural elements which are known as essential
    from other species (template boundary, protein
    binding domains, ?-knot etc.)
  • Novel structural elements
  • Contribute to the general model of the secondary
    and tertiary structure of the yeast telomerase RNA

13
Delimitation of the Gene
  • Gussinova and Tomaska (2007) found the locus of
    TLC1 in six species
  • This locus contains an expected template
    sequence which fits the telomeric repeat
  • The exact limits of the folded sequences were
    determined by multiple alignment against the
    known sequence of C. albicans
  • The Sm protein binding site was found during the
    alignment

14
Secondary Structure Prediction Classical Tools
and Specific Problems of Yeast TER
  • Two basic programs
  • Mfold / RNAfold folds a single sequence to the
    secondary structure with the minimal free energy
    (M. Tzuker)
  • RNAalifold folds a group of aligned sequences.
    Takes into consideration energy, conservation and
    co-variation (Vienna RNA Package)
  • These tools are based on a dynamic programming
    algorithm.
  • The Problems in the case of yeast TER
  • Very long sequences (1500-1800 nt)
    many different
    reasonable optional foldings
  • Ignoring previous biological data (conserved
    sub-sequences and functional structural elements)
  • These tools cannot predict pseudoknots

15
Most of the RNA structural elements obey the
brackets rule
( ( D ) ( A ) ( T ) )
ACGUGCCACGAUUCAACGUGGCACAG ((((((((
))))))))
16
Pseudoknot is a RNA structural element which
breaks the brackets rule
3
j
j
i
i
5
These two stems cross each other. Formally, i j Dynamic programming algorithms cannot predict
such structures!
17
CS3 and CS4 form a special structure of a
pseudoknot which contains a triple helix
Lets convert it to formal constraints
CS3
CS4 (
( ( ( ( ( ( ( ( ( )
) ) ) ) ) ) ) ) )
stem 1 UUU UUU stem 1
AAA
( ( (
( ( (
18
Searching for the Pseudoknot
  • Generate a base pairs matrix for each sequence
  • Represent all optional stable stems (parameters
    length, H. bonds, bulges)
  • Find all optional structures which hold the
    pseudoknots constraints
  • Look for conservation of the pattern at closed
    locations
  • Will the program find the PKs of the known
    species?

19
Pseudoknot - Results
  • The program found the CS3 of Kluyveromyces and
    Saccharomyces as the most likely option.
  • There was found a homologous sequence for the
    Candida species, in an appropriate place within
    the whole sequence.
  • From the relevant candidates for CS4, there is
    one with high similarity to S.cerevisiae, both in
    sequence and structure.

20
Evolution of the CS3 Loop
The stem of CS3 is not conserved at all among the
different groups.
21
Models of the Pseudoknot Saccharomyces,
Kluyveromyces, Candida
K. lactis
22
Est1p Binding Site
obtained by a manipulation of a motif finding
program (MEME), Mfold, RNAalifold, and previous
knowledge
23
An Essential Three-Way Junction
  • Approaches
  • Pattern Location
  • Parsing tree of a probabilistic CFG

24
Future Directions
  • Find all known essential elements (TWJ, template
    boundary etc.)
  • Demonstrate a full secondary structure model
  • Identify additional tertiary interactions
  • Generalize the results to a model that will
    enable genomic searching

25
Acknowledgments
  • Dudy Tzfati
  • Lubo Tomaska, Josef Nosek
  • Nickolay Ulyanov
  • Yogev Brown
  • Hanah Margalit
  • Tommy Kaplan
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