Download Self-Organizing Bio-structures

Self-Organizing Biostructures
NB2-2009
L.Duroux
Lecture 3
Self-Assembly in nucleic acids
DNA & RNA folding
What is RNA?

Aside of being DNA’s “messenger”, RNA
performs functions itself

RNA secondary structure is related to
mRNA stability & RNA functions

RNA folding can be predicted & the
effects of mutations modeled
RNA Primary Structure
(-e)
5'
Structure of
RNA backbone
(-e)
(-e)
(-e)
3'
• RNA chain directionality: 5'3'
• Backbone carries charge (-e) on each nucleotide
• Formation of an RNA structure requires cations
Four Types of Bases
Adenine (A)
Uracil (U)
Guanine (G)
Cytosine (C)
Purines
Pyrimidines
Base-Pairing: a famous case of molecular
self-assembly
A
U
G
C
Watson-Crick canonical base pair
What does RNA do?
The Central Dogma
transcription
splicing
mRNA
tRNA
translation
ribosome
DNA
pre mRNA
mRNA
protein
RNAs are Critical to Cellular Functions

Messenger RNA (mRNA)


Small nuclear RNAs (snRNA)


is the integral part of the
ribosome
Small interfering RNA
(siRNA)


carries amino acid to
ribosome
Ribosomal RNA (rRNA)


splice mRNA in nucleus
Transfer RNA (tRNA)


codes for protein
mRNA turn-over, defense
mechanism
Micro RNA (miRNA)

Gene expression regulation
Some biological functions of non-coding
RNA

snRNA: RNA splicing, telomere maintenance, transcription
regulation

miRNA: translational control (down regulation)

siRNA: RNA interference, gene specific down regulation

Guide RNAs: RNA editing (mitochondria protozoa)

Ribozymes: Catalysis in ribozomes
The function of the RNA molecule depends on its
folded structure
RNA Structure(s)
The RNA Helix
ssRNA forms A-helix:
Grooves
Binding sites
RNA secondary structure
U

Defined by basepairing

Form short helical
structures
Base pairing in RNA: not necessarily
canonical!
Torsion Angles define 3D structure
P
O
c
c
c
O
P
each bond ~ 1.5 Å
nucleotide structure
We need 7 torsional angles per nucleotide to
specify the 3D structure of an RNA
Torsion angles are like rotamers of
protein side chain
RNA specific folds

The RNA molecule folds on itself.

The base pairing is as follows:
G
5’
C
A
U
G
hydrogen bond
3’
GAUCUUGAUC
LOOP
U
UU
C
U
A
G
G
A
U STEM
C
5’
3’
RNA Secondary Structure Motifs
Pseudoknot
Stem
Interior Loop
Single-Stranded
Bulge Loop
Junction (Multiloop)
Hairpin loop
Image– Wuchty
Secondary structure motifs and symbols
Secondary Structure Contact (Base Pair)
Tertiary Structure Contact (Base Pair)
RNA Pseudoknot
Example of RNA tertiary structure: tRNA
RNA Folds &
Function
RIBOZYMES

Catalytic RNA

Can work alone
(Mg2+) or with
proteins

Therapeutic
applications?
Control of iron levels by mRNA
secondary structure
Iron Responsive Element
GU
(IRE) on mRNA
A
G
C
N
N
N’
conserved
N
N’
N
N’
N
N’
C
N
N’
N
N’
N
N’
N
N’
5’ N
N’ 3’
Recognized by Iron
Responsive Protein
(IRP1, IRP2) when Fe
deficiency
F: Ferritin = iron storage
TR: Transferrin receptor = iron uptake
IRP1/2
IRE
F mRNA
3’
5’
IRP1/2
3’
TR mRNA 5’
Low Iron
High Iron
IRE-IRP inhibits translation of Ferritin
IRE-IRP Inhibition of degradation of TR
IRE-IRP off -> Ferritin translated
Transferrin receptor degraded
Structure-based similarity
Sequence Similarity
gurken
Ifactor
%ID = 34%
AAGTAATTTTCGTGCTCTCAACAATTGTCGCCGTCACAGATTGTTGTTCGAGCCGAATCTTACT 64
---TGCACACCTCCCTCGTCACTCTTGATTTT-TCAAGAGCCTTCGATCGAGTAGGTGTGCA-- 58
*
*
***
** ***
***
* * ***** *
*
Structural Similarity
H
H
St
St
Gurken :
(miRNA
controlling
development)
64nt stem loop
I1
I1
B
B
I2
I2
I Factor :
(retrotransposon)
58nt stem loop
RNA Folding & Predictions
RNA folding predictions
Goal: To predict function of an RNA from its
sequence from:
structure
stability
folding kinetics
Ultimate goal: To predict RNA function from its
sequence
Folding Free Energy of Secondary Structure
Folding free energy:
ΔG = G ( secondary structure)
-G(
ΔG = ΔH – T ΔS
)
RNA
PROTEIN
types of sidechains:
4
20
backbone:
7
2
secondary structure:
helices
α, β, ……
# of folded states:
often > 1
usually 1
folding driving force:
base stacking
specific
H, Ф
nonspecific
secondary structure stable without tertiary
stability:
(7bp ~10 kcal/mol)
folding pathway:
electrostatics:
unstable w/t tertiary
(ΔGtot ~10 kcal/mol)
multistate, hierarchical
usually kinetically
controlled
usually 2-state
usually thermodynamically
controlled
highly charged
variable
Applications for RNA folding
predictions




Explain why non-(protein) coding regions
are conserved
Viral RNA packing inside capsid
Prediction of functional RNAs
Identify similarity, not by sequence but by
structure
Why Study RNA Folding Kinetics?
B
A conversion is slow as compared with the translational process
Conformation B is kinetically trapped.
Kinetics is tied to Function
Ion-Dependence of RNA folding
H2O and metal ions are integral
parts of nucleic acid structure
[Na+] stabilizes secondary structure
From Tinoco & Bustamante,JMB (1999) 273,271

[Na+]
by 10 folds
Tm
by 3.8 C
Multivalent Ions Stabilize Tertiary Fold
Pseudoknot
Co(NH 3 )63
[Mg2+] Stabilization
Na+ = 200mM
2
+ 50 M Mg
From Tinoco & Bustamante,JMB (1999) 273,271
RNA conformational changes are iondependent
tRNA
RNA folding kinetics strongly depends
on ions
Na+
Secondary structure
10  100s
Mg2+
10 - 100 ms
for tRNA
Tertiary structure
Metal ion binding sites can be formed before, during, or after the formation
of the tertiary structure
DNA structure
DNA Stabilization--H-bonding
between DNA base pair stacks
Advantages to Double Helix

Stability---protects bases from attack by
H2O soluble compounds and H2O itself.

Provides easy mechanism for replication
Formal geometrical models for
describing shape of Helix

Allows for molecular modeling based on primary
structure

Based on Free-energy computations and
minimization algorithms

Useful to predict impact of sequence
composition or mutations (non-canonical basepairing) on helical structure
Parameters that define base pairs
3DNA (v1.5) — A 3-Dimensional Nucleic Acid
Structure Analysis and Rebuilding Software Package
Xiang-Jun Lu, Wilma K. Olson
Parameters that define sequential base
pair steps
3DNA (v1.5) — A 3-Dimensional Nucleic Acid
Structure Analysis and Rebuilding Software Package
Xiang-Jun Lu, Wilma K. Olson
Parameters that relate base pair to the
helical frame
3DNA (v1.5) — A 3-Dimensional Nucleic Acid
Structure Analysis and Rebuilding Software Package
Xiang-Jun Lu, Wilma K. Olson
Physical Structure (cont’d)


Chains are anti-parallel (i.e in opposite
directions)
Diameter and periodicity are consistent
2.0 nm
 10 bases/ turn
 3.4 nm/ turn


Width consistent because of
pyrimidine/purine pairing
Physical Structure (cont’d)
G-C Content

A=T, G=C, but AT≠GC

Generally GC~50%, but extremely
variable

Examples:
Slime mold~22%
 Mycobacterium~73%


Distribution of GC is not uniform in
genomes
CONSEQUENCES OF GC
CONTENT

GC slightly denser:


Higher GC DNA moves further in a gradient
Higher number of base pairs : more stable
DNA, i.e. the strands don’t separate as
easily.
FORMS OF DNA
DNA forms
B-form
A-form
Z-form
A-DNA vs. B-DNA
A-form
dehydrated
bp/turn
10.9
helical twist angle
33.1°
bp-bp rise
2.9Å
B-form
hydrated
10.0
35.9°
3.4Å
B-DNA is the preferred
conformation in vivo
A “regular” helix contains two
similar grooves
Asymmetric attachment of DNA bases to
backbone creates unequally sized
grooves
Major and minor grooves in
B-DNA and A-DNA
The edges of DNA base pairs can form
hydrogen bonds to protein side chains
Supercoiling
Cruciform
Structures
Another adaptation to supercoiling
Associated with palindromes
DNA is Dynamic


Like proteins, DNA has tertiary structure
Why so many deviations from normal
conformation?
Effects on transcription (gene expression)
 Enhances responsiveness
 May also serve in packaging


NOTE: most cellular DNA exists as protein
containing supercoils
DNA packaging in
chromosomes
Packaging DNA
Histone
octamer
Histone proteins
B DNA Helix
2 nm
Packaging DNA
Histone
octamer
Histone proteins
B DNA Helix
2 nm
Packaging DNA
11 nm
Histone
octomer
Histone proteins
Nucleosome
B DNA Helix
2 nm
Packaging DNA
Packaging DNA
Packaging DNA
“Beads on
a string”
11 nm
30 nm
Tight helical
fiber
Looped
200 nm Domains
Protein scaffold
Packaging DNA
Nucleosomes
11 nm
30 nm
Tight helical fiber
Metaphase
Chromosome
700 nm
200 nm Looped Domains
2 nm
B DNA Helix
Protein scaffold
Chromosomes, Chromatids
and Centromeres
A packaged
chromosome
Chromatid
Identical
chromatid
Chromosome
arm
Centromere
Chromosome
arm
Two identical
chromosomes
Replication
Anaphase
DNA-binding Proteins
Zinc-Finger
Helix-Turn-Helix
Leucine-Zipper
DNA denaturation
Denaturation of DNA


Denaturation by
heating.
How observed?
A260
 For dsDNA,
A260=1.0 for 50 µg/ml
 For ssDNA and RNA
A260=1.0 for 38 µg/ml
 For ss oligos
A260=1.0 for 33 µg/ml
 Hyperchromic shift

The T at which ½ the DNA
sample is denatured is
called the melting
temperature (Tm)
Importance of Tm

Critical importance in any technique that
relies on complementary base pairing
Designing PCR primers
 Southern blots
 Northern blots
 Colony hybridization

Factors Affecting Tm





G-C content of sample
Presence of intercalating agents (anything
that disrupts H-bonds or base stacking)
Salt concentration
pH
Length
Renaturation

Strands can be induced to renature
(anneal) under proper conditions. Factors
to consider:
Temperature
 Salt concentration
 DNA concentration
 Time

Cot Curves
What Do Cot Curves Reveal?



Complexity of DNA sample
Reveals important info about the physical
structure of DNA
Can be used to determine Tm for
techniques that complementary base
pairing.
Complexity of DNA- Factors
Repetitive Sequences


Single Copy Genes
Highly repetitive (hundreds to millions)
Randomly dispersed or in tandem repeats
 Satellite DNA

Microsatellite repeats
 Miniisatellite repeats


Middle repetitive (10- hundreds)
Clustered
 Dispersed


Slightly repetitive (2-10 copies)
Renaturation curves of E. coli and calf DNA
Highly repetitive sequences
Middle repetitive sequences
Unique sequences
The End
Why Study RNA Folding Stability?
Ribosome
binds here
mRNA
mRNA has sufficient time to equilibrate before
translation
is initiated
equilibrium stability
Stability is tied to function
Examples of known interactions of
RNA secondary structural elements
Pseudo-knot
Kissing
hairpins
Hairpin-bulge
contact