Download atomic structure of the DNA double

Lec 3
1) Central Dogma of Molecular Biology always true?
(Wasn’t originally true)
Origin of life was based on RNA; DNA came later.
Solves chicken and egg problem.
2) Biophysics: analyzing via X-
ray crystallography.
Structure of DNA…now structure of
many things.
(Largely due to physicists!)
DNA Making RNA & Proteins
Chicken & Egg Problem
Have already shown that
If DNA is long and therefore very stable. Needs proteins to
replicate itself and to make proteins.
But how can it make the proteins if it needs proteins to make
proteins?
http://lifetech.wiki.hci.edu.sg/DNA+Replication
The answer won a Nobel prize!
Involves RNA…
implications for the start of life on earth.
(Modification to) Central Dogma of Biology
nucleus
nucleus
cytoplasm
cytoplasm
Each time a cell divides, entire DNA gets replicated.
For us, that’s 3 billion base pairs.
Pre-mRNA (or Nuclear RNA)
Copy of DNA with lots of expressed sequences
(exons) and introns (non-expressed sequences)
Present in eukaryotes,
not in “simpler”
bacteria or archea.
http://en.citizendium.org/wiki/Intron
Definition of introns, exons
• http://dictionary.reference.com/browse/intron
• A segment of a gene situated between exons that does not
function in coding for protein synthesis. After transcription of
a gene to [pre-]messenger RNA, the transcriptions of introns
are removed, and the exons are spliced together by enzymes
before translation [into an mRNA] and assembly of amino
acids into proteins.
• Also called intervening sequence.
• http://dictionary.reference.com/browse/exon
• any portion of an interrupted gene that is represented
in the RNA product and is translated into protein.
Example of introns and exons
b-globin gene:
codes for one of two types of
polypeptides making up hemoglobin
In Humans ~ 5% of base pairs in DNA code for a
protein. What is rest for?
Certain fraction does not code for a protein—
rRNA, tRNA…
…read about….
Used to be thought of as “junk” DNA—remnant of
some past when it was useful. Now know that is is
indeed really useful as regulatory RNA.
Known to code for silencing RNA which affects
whether gene is turned on/off.
Alternative Splicing
They can cut out variable # of introns
Adds Complexity
1 Gene, many proteins
Down Syndrome Cell Adhesion Molecule
DSCAM = 1 pre-mRNA = 38,000 potential mRNAs
90% of human genes are alternatively spliced
Splicing
Fundamental for Gene Expression
What role does introns play? Regulation. Read…
Why we’re more complex than just
a tiny worm.
A little worm (19,735 genes, 97 MB), a few mm long
(called C. Elegans) has as many genes as a human
(~21k, 3,000 MB = 3 GB)!
A lot of “genes” are alternatively spliced, can produce
more than one protein.
Until very recently, called DNA that
didn’t code for a protein “junk
DNA”. Now recognize that its very
important.
(Have you read a few articles about
this.)
1 mm____
How are introns/exons cut?
Some by Splicesome; occasionally self-cleavage
Recognize: special sequence.
• Majority cut by Splicesome
(RNA + protein complex).
• Minority self-cleave.
(such as rRNA)
Implications are huge!!
RNA can not only be genetic
information but can also be a
catalyst
RNA solves the chicken & egg problem
From Nobel Lecture
The discovery of catalytic properties in RNA also gives us a new insight into
the way in which biological processes once began on this earth, billions of
years ago. Researchers have wondered which were the first biological
molecules. How could life begin if the DNA molecules of the genetic code
can only be reproduced and deciphered with the aid of protein enzymes, and
proteins can only be produced by means of genetic information from DNA?
Which came first, the chicken or the egg? [Sid] Altman and [Tom] Cech have
now found the missing link. Probably it was the RNA molecule that came
first. This molecule has the properties needed by an original biomolecule,
because it is capable of being both genetic code and enzyme at one and the
same time.
Presentation Speech by Professor Bertil Andersson of the Royal Swedish Academy of
Science, December 10, 1989
RNA can be catalytic!
Life probably started with RNA (not DNA)
1989 Nobel Prize—Altman & Cech
(1967 Carle Woese suggested RNA can be catalytic—won the equivalent of Nobel Prize)
Difference between DNA and RNA
Bases: A:T 2H-bonds; G:C 3H
Backbone:
held together Covalent bonds
Antiparallel:
5’ to 3’ binds to 3’ to 5’
Negative charge on PO4.
(effect of salt)
Sugar: Deoxyribose (2’ = H)
OH
3
1
2
OH
OH
4
OH
5
OH
OH
RNA: Ribose (2’=OH)
Small difference, huge
impact.
Uracil substituted for Thymine
Differences between RNA and DNA
a)
c)
b)
d)
a)
RNA: same nucleotides as DNA, except U substituted for T, the former missing
a methyl group.
b) U is not as particular as T in terms of base pairing, namely it will base-pair
with G. Implications for mutations?
Thymidine greatly improves the efficiency of DNA replication, by reducing the rate of
mismatches, and thus mutations. Also, methylation protects DNA from viruses.
c) RNA is generally single-stranded (-OH doesn’t allow room for dsRNA) but can
form dsRNA—A-form which is more reactive than B-form, which is DNA
structure. Implications for chemical diversity?
(https://en.wikipedia.org/wiki/RNA)
RNA: can attack itself via deprotonation
of 2’ OH
RNA has 2’ OH group.
Under basic conditions 2’ OH becomes O-
Note: DNA has
deoxy (an H)
O- attacks the PO4, cleaves it,
separating the backbone.
RNA  single RNA’s under basic conditions
http://en.wikipedia.org/wiki/RNA
Maybe why RNA isn’t a good lifetimelong storage of genetic information.
But…RNA can be it’s own enzyme!
RNA can be a ribozyme –a ribonucleic acid and enzyme–is an RNA
molecule with a well defined tertiary structure that enables it to
catalyze a chemical reaction. It contains an active site made
completely of RNA. Can cut either itself or another RNA.
http://en.wikipedia.org/wiki/Ribozyme
Used by nature! The ribosome which is used to make proteins
from RNA, is itself a ribozyme (involves RNA cutting by
another RNA).
2009 Nobel Prize (Ramakrishnan, Cambridge; Steitz, Yale; Yonath;
Weizmann)
Evidence that RNA have these properties?
The Ribosome is an RNA-based catalytic machine– Big
surprise! An (r)RNA is used to cut another RNA.
50S subunit of Ribosome
Will discuss more when we go over the Ribosome.
Nice web-site on RNA vs. DNA
http://sites.fas.harvard.edu/~biotext/animations/Ribozymes.swf
X-ray Diffraction
One of the premier techniques of Structural Biology
Can get Angstrom resolution of nearly every atom in biological
macromolecules—now- a-days get up to (beyond?) 2 MD (Ribosomes).
1st one: Whale myoglobin
--won Nobel Prize, 1962, Max
Perutz, Sir John Cowdery
Kendrew.
Watson & Crick 1953
Interbase distance
0.34 nm = P/10
X pattern
Minor Groove
1.2 nm = 3P/8
Period
P = 3.4 nm
Major Groove
2.2 nm = 5P/8
Diameter 2 nm
Fraunhofer X-ray Diffraction
Maximum possible resolution ~λ
X-rays: 10 – 0.1 nm ~Interatomic distances
X-ray Diffraction is elastic scattering
86,817 X-ray crystal structures of proteins, nucleic
acids and other biological molecules have been
determined
The scattering is elastic; the scattered Xrays have the same wavelength as the
incoming X-ray. By contrast, inelastic X-ray
scattering methods are useful in studying
excitations of the sample, rather than the
distribution of its atoms.
“Photo 51” – Rosalind Franklin 1952
The trick is to go from this xray diffraction pattern and
recapture the structure
which leads to this.
X pattern
Layer Lines
Missing 4th layer
Diamond Pattern
4 patterns need to be explained
Sidenote:
What is DNA’s major and minus grooves.
Why different sizes?
a.
b.
c.
10bp/1
turn
(a and b) The 3-D structure of DNA is an anti-parallel double-stranded B-helix,
with a diameter of about 2 nm and 10 base pairs per turn, corresponding to 3.6 A
per base pair. It has a major and minor grove. c) Theoretically, the size of the
major and minor grooves could be anything, from equal (far-right) to a major
difference (left two diagrams). The relative size of the major and minor grove is
determined by the base pair structure.
Class evaluation
1. What was the most interesting thing you learned in class today?
2. What are you confused about?
3. Related to today’s subject, what would you like to know more about?
4. Any helpful comments.
Answer, and turn in at the end of class.