Download Watson, Crick and Wilkins

The Molecular
Structure of DNA
FRANCIS HARRY COMPTON CRICK, JAMES DEWEY WATSON AND MAURICE HUGH FREDERICK WILKINS, 1962
ARTHUR KORNBERG AND SEVERO OCHOA, 1959
ROBERT W. HOLLEY, HAR GOBIND KHORANA AND MARSHALL W. NIRENBERG 1968
WERNER ARBER, DANIEL NATHAN AND HAMILTON O. SMITH, 1978
ANDREW Z. FIRE AND CRAIG C. MELLO, 2006
Francis Harry Compton Crick, James Dewey Watson and
Maurice Hugh Frederick Wilkins, 1962, “for their discoveries
concerning the molecular structure of nucleic acid and its
significance for information transfer in living material”
*
DNA
Deoxyribonucleic acid
*More about Rosalind Franklin later
The Laureates
From left to right:
Maurice Wilkins
Max F. Perutz (Chemistry)
Francis Crick
John Steinbeck (Literature)
James Watson
John C. Kendrew (Chemistry)
Not pictured here:
Linus Pauling (Peace)
Lev Landau (Physics)
James D. Watson
Francis Crick
Maurice Wilkins
Two important players who didn’t get the prize
Rosalind Franklin
Linus Pauling
X-Ray Diffraction
Three forms of DNA
X-ray Diffraction of DNA
DNA: The building blocks
Keto-enol
Tautomers
The cross bridges are pairs made up
of one purine and one pyrimidine
1953: Watson and Crick elucidate the
structure of DNA
http://www.dailymotion.com/video/xitlyu_life-storythe-race-for-the-double-helix-1-2_shortfilms
DNA: basic structure
Arthur Kornberg and Severo Ochoa, 1959, “for
their discovery of the mechanism in the
biological synthesis of ribonucleic acid and
deoxyribonucleic acid”
Kornberg (1918-2007): two major accomplishments (1956):
• He isolated DNA polymerase I, the enzyme that links
nucleotides (deoxyribose + base) through phosphate
linkages
• Using radioisotopes and two phosphodiesterase enzymes
that cleave DNA only from the 3’ end, he showed that
DNA is always synthesized from the 5’ to the 3’ end.
Ochoa (1905-1993) initially studied intermediary metabolism.
He was primarily interested in ATP and enzymes that catalyze
oxidative phosphorylation. Working with Marianne GrunbergManago, he discovered what he thought was RNA
polymerase. Grunberg-Manago felt that the enzyme was in
fact a polynucleotide phosphorylase which breaks down
RNA. She was right, but this was not shown until after the
Nobel prize.
Robert W. Holley, Har Gobin Khorana and Marshall W.
Nirenberg, 1968 “for their interpretation of the genetic
code and its function in protein synthesis”
By 1959 it was accepted that DNA produced RNA which in turn somehow coded for
proteins. What was not known was the length of each DNA codon (George Gamow
theorized 3, as the minimal code for all 20 amino acids) or the actual code itself.
Nirenberg and his post-doc Johann Matthaei used a cell-free E. coli extract with
synthetic RNA and radioactive amino acids to deduce the code and the length of
the codon*.
• First they used repeat polymers such as UUUU… which produced PhePhePhe…
• Next they used mixtures of known ratios of nucleotides to make the RNA: e.g., 3:1 U:G.
• Thus P(UUU)= ¾* ¾ * ¾ , so 27 of every 64 triplets is UUU
• P(UUG)= ¾ * ¾ * ¼, so 9 out of 64 triplets is two U’s and one G
• P(UGG)= ¾ * ¼ * ¼, so 3 our of 64 triplets is one U and two G’s
• Result: VAL, LEU and CYS are incorporated 1/3 as often as PHE, so their codes have two U’s and one G.
But in what order?
• Lastly they bound triplets to ribosomes and added the necessary ingredients. They found the specific Aas
that bound to the triplet-loaded ribosomes
• In the end, they identified 47 of the 64 possible triplets.
*Ochoa was trying much the same thing, in a much better-equipped lab. Since
Nirenberg and Matthaei were working at NIH, many NIH scientists pitched in to help
get a Nobel for the NIH.
Holley, Khorana and Nirenberg, 1968
Khorana, working independently, used a different approach: he
synthesized DNA with only a few bases in sequence, e.g.,
ATGATG…. Using this method Korana produced mRNAs of defined
sequence and thus resolved some ambiguous codes. For example,
mRNA GUGUGUGU---, has two possible codons, GUG or UGU. It
produces CysValCysVal…Knowing that UGU codes for Cys, GUG
must code for Val.
Holley, an organic chemist, was able to determine the structure of
transfer RNA, the RNA which at one end attaches to a specific
amino acid and at the other has a code complementary to that
on the messenger RNA.
Transfer RNA and the
Genetic Code
Werner Arber, Daniel Nathans
and Hamilton O. Smith, 1978 “ for
the discovery of restriction
enzymes and their application to
problems of molecular genetics”
Arber discovered these enzymes in the early 1960s when he analyzed an apparently obscure
phenomenon in bacteria, discovered 10 years earlier by Bertani and Weigle, called host-controlled
modification. Arber showed that this phenomenon was caused by a change in DNA and apparently
served to degrade Foreign DNA. Arber postulated that bacteria contain enzymes with the capacity to
recognize and bind to precise sequences in the DNA and sever it at those loci.
Smith verified Arber's hypothesis. He purified one restriction enzyme and showed that it could cleave
foreign DNA. He determined the chemical structure of the regions of DNA which were severed by the
enzyme and discovered certain rules which later could be applied to other restriction enzymes.
Nathans pioneered the application of restriction enzymes in genetics. He constructed the first genetic
map using restriction enzymes by cleaving the DNA from a monkey virus. The methodology devised by
him for this purpose was later used by others to construct increasingly more complicated maps.
Plasmids and Restriction
Enzymes
Andrew Z. Fire and Craig C. Mello, 2006, “for their
discovery of RNA interference-gene silencing by
double-stranded RNA”
Fire and Mello in 1998* found that if they injected fragments of
double-stranded RNA (dsRNA) into C. elegans, they could
selectively turn off certain genes if one strand of the dsRNA
was complementary to the gene on the DNA.
We now know that such exogenous dsRNA, or RNAi, uses an
ancient mechanism for regulating gene expression using micro
RNA (miRNA) produced by the cell itself. Essentially, RNAi
mimics miRNA.
miRNA is produced in the nucleus. After some processing there it moves into the cytoplasm. There it is
cleaved by an enzyme called Dicer, and is incorporated into a complex called RISC (RNA-Induced
Silencing Complex), which binds to messenger RNA (mRNA) and modulates expression of that mRNA
into proteins.
Introduced dsRNA differs from miRNA in that it forms a RISC which is much more specific for the
mRNA sequence of its target.
*Fire, et al., “ Potent and specific genetic interference by double-stranded RNA in
Caenorhabditis elegans” Nature 391, 806, 1998)
Interference RNA
Why natural RNAi? Two good reasons for this defense
mechanism:
• Viruses
• Some viruses are dsRNA viruses (e.g., rotavirus which
causes gastroenteritis in children, and picobirnaviruses
that cause diarrhea).
• This dsRNA could trigger the RNAi system and lead to the
suppression of virally-induced mRNA
• Jumping genes
• Many transposons are delimited by terminal inverted
repeats, which could potentially give rise to dsRNA which
in turn could trigger the RNAi system.
Viral mRNA

Animation of RNAi Mechanism
Therapeutic and Research uses of RNAi

Therapy:

Dominant Negative Mutations:


One allele produces an abnormal protein which outcompetes the normal protein for the
appropriate receptor. Many of these mutations are SNPs. Drugs cannot differentiate between the
normal and mutant protein, but RNAi might be able to target and degrade the mutant mRNA
Viral Diseases:

Introduce a dsRNA (or an artificial DNA that transcribes into a dsRNA (hairpin) which is
complementary for the viral mRNA.


Cancer:


Currently clinical trials are underway for among other things hepatitis C and HIV.
RNAi could be used to knock out cancer-producing genes, or perhaps even to upregulate
cancer-suppressing ones.
Research:

Learn the function of a gene by blocking its expression.
Delivery of
RNAi
In any therapeutic use of
RNAi the difficulty is to
get the RNAi to its target
cells.
This includes
preservation in the
bloodstream, prevention
of immune response,
and entry into the
specific target cell.
The CRISPR-CAS 9 System: Next
Nobel Prize??
Fig. 1 Overview of the four CRISPR/cas
systems present in Streptococcus
thermophilus DGCC7710.
Fig. 2 Overview of the CRISPR/Cas
mechanism of action.
Philippe Horvath, and Rodolphe Barrangou Science
2010;327:167-170
Published by AAAS
Fig. 3 CRISPR interference.
Philippe Horvath, and Rodolphe Barrangou Science
2010;327:167-170
Published by AAAS
DNA surgeon.
With just a guide RNA and a protein
called Cas9, researchers first showed
that the CRISPR system can home in on
and cut specific DNA, knocking out a
gene or enabling part of it to be
replaced by substitute DNA. More
recently, Cas9 modifications have
made possible the repression (lower
left) or activation (lower right) of
specific genes.
Published by AAAS
Elizabeth Pennisi Science 2013;341:833-836
Editas Medicine, Inc.