Molecular Genetics
Nucleic Acids
http://learn.genetics.utah.edu/
Learn Genetics
University of Utah Genetics Learning Center
Ch 12,13,15
The Central
Dogma of
Molecular
Biology
DNA
RNA
Protein Synthesis
1957…Crick
Milestones in DNA History Test
Text- p263- Table 12-1
• 1869/1871 Johann Friedrich Miescher identifies a weakly acidic substance of
unknown function in the nuclei of human white blood cells. This substance will
later be called deoxyribonucleic acid, or DNA.
(Nuclein = DNA, RNA, Proteins)
• *what they knew: Pasteur 1862…germ theory, No ‘spontaneous generation’
Mendel ‘rediscovered’ 1900’s ……. Fleming 1928…penicillin Vaccines/Meds…
• *genes stored genetic information…Proteins/20 different amino acids,
’DNA’/simple base unit….so….proteins must store the genes.
Milestones in DNA History
• 1928 Franklin Griffith, a British medical officer, discovers that
genetic information can be transferred from heat-killed bacteria
cells to live ones. This phenomenon, called transformation,
provides the first evidence that the genetic material is a heatstable chemical.
(Text-p261)
*Griffith was trying to develop a vaccine against pneumonia when
he serendipitously discovered the phenomenon of transformation.
….scientists hypothesized that a chemical substance (a transforming
principle) was transferred from the dead bacteria to the living cells
and caused transformation
Griffiths experiments- 1928
• Textp261
Although neither
the rough strain
nor the heatkilled strain
smooth strain
could kill a
mouse, a combo
of the two did.
Autopsy showed
the presence of
living S-strain
cells in the dead
mice…results
indicated that
some substance
in the heat-killed
S cells had
transformed the
living R cells into
a virulent form.
“transforming principle”
Milestones in DNA History
• 1944 Oswald Avery, Maclyn McCarty and Colin MacLeod, identify
Griffith's transforming agent as DNA. Their discovery is greeted
with skepticism, in part because many scientists still believe that
DNA is too simple a molecule to be the genetic material.
By lysing S cells, separating
the contents- lipids, proteins,
polysaccharides, nucleic acids
(DNA,RNA) and testing each
fraction to see if it could
transform living R cells into
S cells. Only when R cells
were treated with the nucleic
acids from the S cells were
they transformed.
…still…it’s the protein
Milestones in DNA History
• 1952: Alfred Hershey and Martha
Chase
(Text-p 262)
• Demonstrated that DNA, not
protein, is involved in viral
reproduction.
The “transforming agent”
• Tagged the protein coat of one
bacteriophage (virus that infects
bacteria) with one radioactive
isotope, and the viral DNA of a
second sample with a different
isotope (red)
• Infected, Blended, centrifuged:
protein still outside the cell, DNA in
the portion with the bacterial cells
Race is on….
...Discovery of the structure of DNA?
Milestones in DNA History
The Necessary Groundwork….
• 1912 Physicist Sir William Henry Bragg, and his son, Sir William
Lawrence Bragg, discover that they can deduce the atomic
structure of crystals from their X-ray diffraction patterns. This
scientific tool will be key in helping Watson and Crick determine
DNA's structure.
• “Crystallography”
(Text p 265)
Milestones in DNA History
The Necessary Groundwork….
• 1952 Maurice Wilkins and Rosalind
Franklin/ Franklin and Raymond Gosling:
work on X-ray diffraction experiments on
DNA. Thanks largely to Franklin’s
expertise in crystallography - the vital
“photo 51” was taken in May 1952.
• Watson and Crick cracked the structure
of DNA in March 1953. The spur for Jim
Watson's new attempt at model building
had been seeing the clear helical pattern
of "photo 51" and deciding to discard
experimental data pointing to three
chains and opt for two.
Milestones in DNA History
The Necessary Groundwork….
• 1949 Erwin Chargaff: Biochemist. Reports that the composition of
DNA is specific; amount of adenine equals the amount of thymine
and the amount of guanine equals the amount of cytosine in DNA
from EVERY species………..Helix becomes a double helix
Milestones in DNA History
• 1953 James Watson and Francis
Crick discover the molecular
structure of DNA.
Compiled data/made model
“The forest through the trees”
• 1962 Francis Crick, James Watson,
and Maurice Wilkins receive the
Nobel Prize for determining the
molecular structure of DNA.
Great Discoveries in Science: The Double Helix 16.53
http://www.ted.com/talks/james_watson_on_how_he_discovered_dna.html
~14 minutes
"This structure has novel features which are of considerable biological interest"
DNA vs RNA
• Double Helix vs Single strand
• DNA- deoxyribose sugar
(one less O than ribose)
RNA- Ribose sugar
• 4 Nitrogen Bases
DNA:
RNA:
A-T
C-G
A-U
C-G
• DNA stays in the nucleus,
RNA made in the nucleus,
works in the cytoplasm…
where?
• DNA contains information to
direct development;
RNA: 3 main types carry
out DNA directions:
mRNA = messenger
tRNA = transfer
rRNA = ribosomal
Structure of DNA
• DNA is made up of nucleotides, each consisting of:
• A sugar “deoxyribose”
• A phosphate group
• A nitrogenous base:
• Adenine=Thymine
• Cytosine =Guanine
• Covalently Bonded: “the brick”
• # Carbons?
O
5
1
4
3
2
DNA at the molecular level
C3’ (OH Group)
3’ end
Hydrogen Bonds
C5’
5’ prime end (Phosphate Group)
C1’
(Nitrogenous base)
Anti-parallel Orientation of DNA
BUILD
READ
• 5’ end:
Phosphate
group
• 3’ end: OH
group
• Antiparallel
• Hydrogen
Bonding
betweenindividually
weak,
collectively
strong
• Built in a
5’ to 3’ direction,
Read 3’ to 5’
Note: Difference in shape
between A, G and T,C?
Purines- 2 rings, Pyrimidines- 1ring
Purine bonds with a Pyrimidine (3ring)
Structure of DNA
• http://www.hhmi.org/biointeractive/disease/dna_chem_struc
ture.html 2.44
(play Large WMV)
Semiconservative Replication
TEXT p268
• One strand of parent in EACH new strand
• Explains the perpetuation of mutations
Meselson-Stahl Experiment 1958 Disproves Conservative (b), dispersive (c)
DNA
Replication
Replication Fork
-Happening at many places
along the molecule
DNA Replication Factory
Replication in action 7,8
VIDEO: REPLICATION ENZYMES
DNA Replication:
Replicating enzymes- Unwinding of DNA
1. Initiator Protein: recognizes where to begin
2. Helicase Enzyme: Lands at the origin- starts
breaking the Hydrogen bonds between the bases,
*working in both directions, at the replication fork
3. SSBP’s (Single Stranded Binding Proteins)-keep the
strands from joining again
4. Primase Enzyme: the RNA “primer”/marker on the
open end- marks where to begin adding
nucleotides
5. DNA Polymerase: adds complimentary
nucleotides, one at a time
(A to T, G to C)
DNA Replication:
Replicating the strands: Leading vs Lagging
Replication Bubble
VIDEO: REPLICATING THE STRANDS
DNA Replication:
Replicating the strands: Leading vs Lagging
LEADING STRAND: Builds towards the replication fork
in a continuous manner, reading 3’ to 5’ and building
5’ to 3’
LAGGING STRAND: Builds away from the replication
fork in a discontinuous manner creating, using DNA
primase at each starting point.
OKAZAKI FRAGMENTS –segments of DNA
DNA LIGASE- welds together the okazaki
fragments into a continuous strand.
DNA Replication:
The Twisting Problem
TOPOISOMERASE: Enzyme that prevents kinking of
the DNA strand as it unwinds
VIDEO: THE TWISTING PROBLEM
DNA
Replication
Proofreading
and Repair
“Enzymes on Patrol”
• Reduces the rate of
error to ~ 1/billion
base pairs
• Mutations:
harmful, fatal or
just part of the
evolutionary
process
VIDEO: PROOFREADING and REPAIR
Backs up, replaces with correct base
RNA SYNTHESIS
• Messenger RNA (mRNA)
• Single, uncoiled chain
• Carries genetic information from the nucleus to the
ribosomes
• Transfer RNA (tRNA)
• Single chain folded into a hairpin shape
• Carries amino acids from the cytoplasm to the
ribosomes
• Ribosomal RNA (rRNA)
• Globular
• Found in the ribosome and assists in protein synthesis
HANDOUT: How DNA makes RNA
THE
MAKING
OF
A
PROTEIN
1. Transcription- mRNA
Process of copying DNA into mRNA:
Every 3 bases will code for 1 amino acid…..building a protein
• Transcription
(animation-next concept)
http://www.phschool.com/science/biology_place/biocoach/transcription/tcproc.html
Video: Main Menu>Genetic Code & Protein Synthesis>RNA>Transcription
2. Translation- tRNA
*DNA base
sequence (triplets)
of the “gene”
coding for the
synthesis of a
particular
polypeptide chain.
*Base sequence
(codons) of the
transcribed mRNA
*Consecutive base
sequences of tRNA
anticodons
capable of
recognizing the
mRNA codons
“calling” for the
amino acids they
transport
*Translation BEGINS when mRNA LEAVES the nucleus
2. Translation- tRNA
• Begins once mRNA leaves the nucleus.
• Ribosomes in the cytosol make proteins for the cell;
• Ribosomes on the rough ER make proteins for export.
The Genetic Code: Specific for Amino Acids
DNA:
Triplets
TAC
mRNA:
Codons
AUG
tRNA:
Anticodons UAC
= Amino Acid:
Methionine (start)
**The CODON determines the Amino Acid NOT the anticodon
•
•
# of different Amino Acids?
Possible Code Combinations?
Ribosomal Subunits,
mRNA and tRNA
• Large
Ribosomal
subunit
• Small
Ribosomal
subunit
• P site
• A Site
• tRNA: AA
attachment
site;
anticodon
Translation
1. Codon recognition:
translation begins
2. Peptide Bond formed
between the amino
acids
3. Translocation:
Ribosome moves
AHEAD; what was on
the P site is now on
the growing peptide
chain, the tRNA on
the A site moves to
the Psite, making
room for the next AA
4. Completing the
protein: Stop codonprotein is released
Video: Main Menu>The Genetic Code & Protein Synthesis>Translation, Protein Synthesis
Codons:
To find:
ACG
“First position”-letter
gives you the correct
row.
“Second position” –
letter gives you the
correct column
“Third position” –final
row
Codon for Threonine
• Ribosome
shifts one
codon in the
3’ direction
• Stop codon:
no anticodon
TRANSLATION
http://bcove.me/lz405t1m 2.06
Summary
Worksheet
The mRNA Editing Process
• A strand of mRNA inside the
nucleus is much longer than the
segment that actually leaves. It is
made up of two parts:
• Exons: Proteins Coding Region of a
Eukaryotic Gene. Exons are the
part that exits the nucleus.
• Introns: Non-protein Coding
Regions of a Eukaryotic GeneIntrons are the part that remain
inside the nucleus. Usually longer.
Thought to be “junk DNA”, now
know this “silent DNA” is
important in gene expression
Transcribed mRNA
in the nucleus
Video: Main Menu>The Genetic Code & Protein Synthesis>Exons and Introns
Exons and Introns
The mRNA Editing Process
• Once the mRNA strand is ready to leave the nucleus (only Exons), a
cap and a tail are placed on the ends of the mRNA to prevent the
genes from being damaged
• All mRNA are read in a 5’ to 3’ direction
• G Cap: (Guanine)- placed at the 5’ end of the RNA
• Poly A Tail: (Adenine)- placed at the 3’ end of the RNA
Mutations
• Random mistake (“spontaneous”) made in the DNA copying process;
not planned, not predictable
• Even with the “Enzymes on Patrol” errors can still occur
• Causes:?......
• UV, drugs, alcohol, X-Ray, chemicals: (“Mutagens”), viruses
• Some are very harmful…where the mutation occurs on the DNA chain
can change its effect. Depends on when occurs in formation of the
zygote, through the adult organism
• Basic Types: Base/Point and Frameshift Mutations
*Substitutions
*Insertions
*Deletions
Video: Main Menu>The Genetic Code & Protein Synthesis>Mutations
• Silent mutations code for the same amino acid.
• Nonsense mutations code for a stop codon, which may cause a shortened protein.
Depending on the extent of protein loss, protein function may be affected.
• Missense mutations:
• Conservative mutations cause a change in amino acid but no change in properties, as
compared to the wild-type.
• Non-conservative mutations induce different properties compared to the wild type. T
The gly → valine transition resulting in a protooncogene → oncogene transition is an
example of this type.
2. Point Mutations
• Substitute one nucleotide for another
• Usually not very harmful
• Ex:
THE CAT ATE THE RAT
THE CAT ATE THE NAT
3. Frameshift Mutation
• One or two nucleotide pairs are inserted or
deleted from the molecule, altering the reading
frame
• Very harmful
Ex: THE CAT ATE THE RAT
……….CA Deleted
THE TAT ETH ENA T
Gene Regulation: Operons
Text p 307
• Two Examples of Regulatory Systems:
1. Inducible- usually in the ‘off’ position
2. Repressible- usually in the ‘on’ position
…Looking at one example of each
• Operons: functionally related genes
regulated together in gene complexes.
1960’s Ecoli Bacteria
The lac operon
“inducible system”
Controlled by
a repressor
keeping it in
the ‘off
position
• Found in prokaryotic cells
• Regulates synthesis of inducible enzymes
• Usually codes for enzymes in a catabolic pathways
• Usually in the ‘off’ position
The lac operon
*
“inducible system”
*Noncompetetive site on the enzyme?
• LACTOSE PRESENT: Repressor is inactive; the operon is ON.
• Enzymes made to break down lactose
• Helps cell save the energy costs of making enzymes when
no substrates (like lactose) are available
Video: Main Menu>The Genetic Code & Protein Synthesis>Gene Regulation in Prokaryotes
Trp operon “repressible system”
”
p310
*Tryptophan- an
amino acid
bacteria need
most of the time
*Anabolic
pathways
*Usually ON