Download AP Molecular Genetics

DNA
The Genetic Material
Chargaff
 DNA composition: “Chargaff’s rules”
varies from species to species
 all 4 bases not in equal quantity
 bases present in characteristic ratio

 humans:
A = 30.9%
T = 29.4%
G = 19.9%
C = 19.8%
That’s interesting!
What do you notice?
Rules
A = T
C = G
1947
1953 | 1962
Structure of DNA
 Watson & Crick

developed double helix model of DNA
 other leading scientists working on question:
 Rosalind Franklin
 Maurice Wilkins
 Linus Pauling
Franklin
Wilkins
Pauling
1953 article in Nature
Watson and Crick
Watson
Crick
Rosalind Franklin (1920-1958)
Discussion
 Summarize: What do you remember
about the chemical composition of
DNA? Consider the following vocab
words?

Nucleotide, hydrogen bond, double
helix, deoxyribose, phosphate,
nitrogenous base, adenine, cytosine,
guanine, thymine, purine, pyramidine,
phosphodiester bond
Double helix structure of DNA
“It has not escaped our notice that the specific pairing we have postulated
immediately suggests a possible copying mechanism for the genetic
material.”
Watson & Crick
Base pairing in DNA
 Purines
adenine (A)
 guanine (G)

 Pyrimidines
thymine (T)
 cytosine (C)

 Pairing

A:T
 2 bonds

C:G
 3 bonds
Directionality of DNA
 You need to
PO4
nucleotide
number the
carbons!

Think Five =
Phosphate
N base
5 CH2
This will be
IMPORTANT!!
O
4
1
ribose
3
OH
2
Anti-parallel strands
 Nucleotides in DNA
backbone are bonded from
phosphate to sugar between
3 & 5 carbons



DNA molecule has
“direction”
complementary strand runs
in opposite direction
3’ and 5’ will determine
where replication and
transcription can begin and
end
5
3
3
5
Bonding in DNA
5
hydrogen
bonds
3
covalent
phosphodiester
bonds
3
5
….strong or weak bonds?
How do the bonds fit the mechanism for copying DNA?
DNA Organization
 DNA is organized in long
strands called
chromosomes.
Circular in prokaryotes
 Linear in eukaryotes

CHECKPOINT
 Without notes, try to diagram or
describe the structure of a strand of
DNA, labeling all molecules, bonds, 3’
and 5’ ends.

If you can’t, memorizing that structure
is your homework tonight!
DNA Replication
2007-2008
But how is DNA copied?
 Replication of DNA
Ensures the
continuity of
genetic information
 base pairing
means each side
will serve as a
template for a new
strand

Copying DNA
 Replication of DNA

new strand is 1/2
parent template &
1/2 new DNA
 = semi-conservative
copy process
DNA Replication
Let’s meet
the team…
 Large team of enzymes coordinates replication
Replication: 1st step
 Unwind DNA

helicase enzyme
 unwinds part of DNA helix (hence “helicase,”
AMAZING I KNOW)
 stabilized by single-stranded binding proteins
helicase
single-stranded binding proteins
replication fork
Replication Fork
 Replication begins at a point on the
chromosome called the “origin.”
 Helicase bonds to the origin, starts
unzipping the strands, and moves
progressively away, forming a
“replication fork.”
helicase
Replication: 2nd step
 Build daughter DNA
strand
add new
complementary bases
 Polymerization, an
anabolic process
 DNA polymerase III

DNA
Polymerase III
But…
But where’s the
We’re missing
ENERGY
something!
for the bonding!
What?
Energy of Replication
Where does energy for bonding usually come from?
We come
with our own
energy!
You
remember
ATP!
Are there
otherenergy
ways
other
to
get energy
nucleotides?
out
it?
You of
bet!
ATP
GTP
CTP
TTP
modified nucleotide
And we
leave behind a
nucleotide!
energy
energy
CMP
TMP
GMP
AMP
ADP
Energy of Replication
 The nucleotides arrive as nucleosides

DNA bases with P–P–P
 P-P-P = energy for bonding


DNA bases arrive with their own energy source
for bonding
bonded by enzyme: DNA polymerase III
ATP
GTP
TTP
CTP
5
Replication
 Adding bases

can only add
nucleotides to
3 end of a growing
DNA strand
 need a “starter”
nucleotide to
bond to

strand only grows
53
B.Y.O. ENERGY!
The energy rules
the process
3
energy
DNA
Polymerase III
energy
DNA
Polymerase III
energy
DNA
Polymerase III
DNA
Polymerase III
energy
3
5
Discussion
 So we follow helicase along and
replicate the strand in the 5’->3’
direction (that’s 5’->3’ of the strand
being build, the template runs 3’->5’
because DNA is antiparallel)… But what
is the problem that we have now
created?
5
3
5
need “primer” bases to add on to
3
energy
no energy
to bond

energy
energy
energy
energy
ligase
energy
energy
3
5
3
5
Okazaki
Leading & Lagging strands
Limits of DNA polymerase III

can only build onto 3 end of
an existing DNA strand
5
3
5
3
5
3
5
5
5
Lagging strand
ligase
growing
3
replication fork
Leading strand
3
Lagging strand

Okazaki fragments



3
Short DNA fragments
joined by ligase
 “spot welder” enzyme

5
3
DNA polymerase III
Leading strand

continuous synthesis
Replication fork / Replication bubble
3
5
5
3
DNA polymerase III
leading strand
5
3
3
5
3
5
5
5
3
lagging strand
3
5
3
5
lagging strand
5
5
leading strand
growing
replication fork 5
3
growing
replication fork
leading strand
3
lagging strand
5 5
5
5
3
Starting DNA synthesis: RNA primers
But there’s yet another
problem!

5
can only build onto 3 end of
an existing strand
3
3
5
5
3
5
3
5
growing
3
replication fork
DNA polymerase III
primase
RNA 5
RNA primer


built by primase, serves as starter
sequence for DNA polymerase III
@ start of leading strand, and at start of
each Okazaki fragment
3
Replacing RNA primers with DNA
Ligase
DNA polymerase I


removes sections of RNA
primer and replaces with
DNA nucleotides
3
5
5
DNA polymerase I
Connects
strands
5
3
ligase
growing
3
replication fork
RNA
5
3
But DNA polymerase I still can only build
onto 3 end of an existing DNA strand. One
primer can’t be acted upon…
Chromosome erosion
Houston, we
have a problem!
DNA polymerase I
5
3
3
5
5
growing
3
replication fork
DNA polymerase III
RNA
Loss of bases at 5 ends
in every replication


chromosomes get shorter with each replication
limit to number of cell divisions
5
3
Telomeres
Repeating, non-coding sequences at the end
of chromosomes = protective cap to erode
instead of gene sequence
5
3
3
5
5
growing
3
replication fork
Telomerase



enzyme extends telomeres
can add DNA bases at 5 end
different level of activity in different cells
 high in stem cells & cancers -- Why?
telomerase
5
TTAAGGG TTAAGGG 3
Replication fork
DNA
polymerase III
lagging strand
DNA
polymerase I
5’
3’
ligase
primase
Okazaki
fragments
5’
3’
5’
SSB
3’
helicase
DNA
polymerase III
5’
3’
leading strand
direction of replication
SSB = single-stranded binding proteins
http://highered.mcgraw-hill.com/sites/0072943696/student_view0/chapter3/animation__dna_replication__quiz_1_.html
Discussion
 Summarize the functions of the DNA
replication enzymes…
Helicase
 DNA polymerase III
 DNA polymerase I
 Primase
 Ligase
 Telomerase

DNA polymerases
 DNA polymerase III
1000 bases/second!
 main DNA builder

 DNA polymerase I
20 bases/second
 editing, repair & primer removal

DNA polymerase III
enzyme
Editing & proofreading DNA
 1000 bases/second =
lots of typos!
 DNA polymerase I

proofreads & corrects
typos

repairs mismatched bases

removes abnormal bases
 repairs damage
throughout life

reduces error rate from
1 in 10,000 to
1 in 100 million bases
Fast & accurate!
 It takes E. coli <1 hour to copy
5 million base pairs in its single chromosome

divide to form 2 identical daughter cells
 Human cell copies its 6 billion bases & divide into
daughter cells in only few hours




remarkably accurate
only ~1 error per 100 million bases
~30 errors per cell cycle
These errors = mutations, can change the type or
amount of protein produced. More on that later…
From Gene
to Protein
How Genes
Work
What do genes code for?
 How does DNA code for cells & bodies?

DNA
how are cells and bodies made from the
instructions in DNA
proteins
cells
bodies
The “Central Dogma”
 Flow of genetic information in a cell

How do we move information from DNA to proteins?
DNA
replication
RNA
protein
DNA gets
all the glory,
but proteins do
all the work!
trait
Transcription
from
DNA nucleic acid language
to
RNA nucleic acid language
RNA as opposed to DNA
 ribose sugar
 N-bases

uracil instead of thymine
U:A
 single stranded
 lots of RNAs

DNA
mRNA, tRNA, rRNA…
transcription
RNA
Kinds of RNA
 The sequence of RNA bases and structure of
the RNA molecule determines its function
 There are more than 100 kinds! Major ones:




mRNA - transcription product, carries info
from DNA to ribosome
tRNA - translation intermediate, converts
genetic info to protein sequence
rRNA - makes up ribosomes
RNAi - various RNA molecules interfere with
transcription, helping control gene expression
Transcription
 Making mRNA


transcribed DNA strand = template strand
untranscribed DNA strand = coding strand
 same sequence as RNA

synthesis of complementary RNA strand
 transcription bubble

enzyme
coding strand
 RNA polymerase or RNAP
5
C
DNA
G
3
A
G
T
A T C
T A
A G C
A
T
C G T
A
C
T
3
G C A U C G U
C
G T A G C A
T
T
A
C
A G
C T
G
A
T
A
T
3
5
unwinding
rewinding
mRNA
build RNA 53
G
5
RNA polymerase
template strand
How does RNAP “know” where to “read?”
 Promoter region



RNAP binding site before beginning of gene
“Tells RNAP to start here”
Many promoters include TATA box binding site
 DNA sequence TATAAA
 Enhancer region

binding site far
upstream of gene
 turns transcription
on HIGH
Transcription Factors
 Initiation complex

transcription factors bind to promoter region
 suite of proteins which bind to DNA
 turn on or off transcription

trigger the binding of RNA polymerase to DNA
http://www.youtube.com/watch?v=41_Ne5mS2ls
http://highered.mcgraw-hill.com/sites/0072507470/student_view0/chapter3/animation__mrna_synthesis__transcription___quiz_1_.html
Matching bases of DNA & RNA
 Match RNA bases to DNA
C
G
bases on one of the DNA
strands
U
A
G
G
U
U
C
A
AG
A
C
G
A
U
A
C
5'
RNA
A C C polymerase G
A
U
3'
T G G T A C A G C T A G T C A T CG T A C CG T
U
C
Eukaryotic genes have junk!
 Eukaryotic genes are not continuous

exons = the “real gene”
 expressed / coding

introns
come out!
introns = the “junk”
 inbetween sequence
intron = noncoding (inbetween) sequence
exon = coding (expressed) sequence
mRNA splicing
 Post-transcriptional processing




eukaryotic mRNA needs work after transcription
primary transcript = pre-mRNA
mRNA splicing
 edit out introns
make mature mRNA transcript
intron = noncoding (inbetween) sequence
~10,000 base
eukaryotic DNA
exon = coding (expressed) sequence
pre-mRNA
primary mRNA
transcript
mature mRNA
transcript
~1,000 base
spliced mRNA
RNA splicing enzymes
 snRNPs


small nuclear RNA
exon
proteins
 Spliceosome


snRNPs
snRNA
intron
exon
5'
several snRNPs
recognize splice
site sequence
3'
spliceosome
5'
3'
 cut & paste gene
No,
not smurfs!
“snurps”
mature mRNA
lariat
5'
exon
5'
3'
exon
3'
excised
intron
mRNA splicing
 Introns are NOT useless junk!


Introns can be “mobile elements,” spliced out of
one gene that then go insert themselves
somewhere else!
More famously, there’s “alternative splicing.” The
exact same introns are NOT excised out of the
mRNA gene sequence every time it is made.
intron = noncoding (inbetween) sequence
~10,000 base
eukaryotic DNA
exon = coding (expressed) sequence
pre-mRNA
primary mRNA
transcript
mature mRNA
transcript
~1,000 base
spliced mRNA
Alternative splicing
 Alternative mRNAs produced from same gene


different segments treated as exons
one gene can thus make multiple proteins
Starting to get
hard to
define a gene!
Discussion
 How can the fact that cells conduct
post-transcriptional processing
enhance genetic diversity? How could
this affect the rate at which traits can
evolve/emerge?
More post-transcriptional processing
 Need to protect mRNA on its trip from
nucleus to cytoplasm

enzymes in cytoplasm tend to attack mRNA
 protect the ends of the molecule
 add 5 GTP cap
 add poly-A tail
 longer tail, mRNA lasts longer: produces more protein
3'
mRNA
5'
P
G P
P
A
Translation
from
nucleic acid language
to
amino acid language
How does mRNA code for proteins?
DNA
TACGCACATTTACGTACGCGG
4 ATCG
mRNA
4 AUCG
protein
AUGCGUGUAAAUGCAUGCGCC
?
Met Arg Val Asn Ala Cys Ala
20
How can you code for 20 amino acids
with only 4 nucleotide bases (A,U,G,C)?
mRNA codes for proteins in triplets
DNA
TACGCACATTTACGTACGCGG
codon
mRNA
mRNA
AUGCGUGUAAAUGCAUGCGCC
AUGCGUGUAAAUGCAUGCGCC
?
Met Arg
protein
Val
Asn
Ala Cys
Cracking the code
 Francis Crick
determined 3-letter (triplet) codon system

 Codon = 3 mRNA bases that will match to 1 amino acid
WHYDIDTHEREDBATEATTHEFATRAT
 Marshall Nirenberg & Har Gobind Khorana
determined mRNA–amino acid match
 added fabricated mRNA to test tube of
ribosomes, tRNA & amino acids

 created artificial UUUUU… mRNA
 found that UUU coded for phenylalanine
The code
 Code for ALL life!

Highly conserved common origin for
all life
 Code is redundant


several codons for
each amino acid
3rd base “wobble”
Why is the
wobble good?
 Start codon


AUG
methionine
 Stop codons

UGA, UAA, UAG
Evolution of the
Genetic Code
 The genetic code is nearly
universal, shared by all living
organisms
 DNA can be transcribed and
translated from one species to
another

Examples:
 Glowing organisms!
 Bacteria making human proteins
How are the codons matched to
amino acids?
DNA
mRNA
3
5
5
3
TACGCACATTTACGTACGCGG
AUGCGUGUAAAUGCAUGCGCC
3
UAC
tRNA
amino
acid
Met
codon
5
GCA
Arg
CAU
Val
anti-codon
Transfer RNA structure
 “Clover leaf” structure
anticodon on “clover leaf” end
 amino acid attached on 3 end

Ribosomes
 Facilitate coupling of
tRNA anticodon to
mRNA codon
 Structure
ribosomal RNA (rRNA) & proteins
 2 subunits

 large
 small
E P A
Ribosomes
 A site (aminoacyl-tRNA site)

holds tRNA carrying next amino acid to
be added to chain
 P site (peptidyl-tRNA site)

holds tRNA carrying growing
polypeptide chain
Met
 E site (exit site)

empty tRNA
leaves ribosome
from exit site
U A C
A U G
5'
E
P
A
3'
http://www-class.unl.edu/biochem/gp2/m_biology/animation/gene/gene_a3.html
http://www.dnatube.com/video/5934/Basic-explanation-of-mRNA-Translation
http://highered.mcgraw-hill.com/sites/0072507470/student_view0/chapter3/animation__protein_synthesis__quiz_3_.html
Building a polypeptide
 Initiation

brings together mRNA, ribosome
subunits, initiator tRNA
 Elongation

adding amino acids based on
codon sequence
 Termination

3 2 1
end codon
Leu
Val
Met
Met
Met
Met Leu
Ala
Leu
Leu
release
factor
Ser
Trp
tRNA
U AC
5'
C UGAA U
mRNA A U G
3'
E P A
5'
UAC GAC
A U G C U GAA U
5'
3'
U A C GA C
A U G C U G AAU
5'
3'
U AC G A C
AA U
AU G C U G
3'
A CC
U GG U A A
3'

Amino acid sequence yields
function
Proteins have 4 levels of structure




Primary: sequence of amino acids
Secondary: Hydrogen bonds between backbone
causes α-helix or β-pleated sheet
Tertiary: R group interactions causes 3D shape
Quaternary: multiple folded polypeptide subunits
or domains join together
Discussion
 Use one of the genetic codes provided to

transcribe and translate this DNA template
strand.
3’TATAAACCTACGTCGGATCGACGATCGTAG5’
RNA polymerase
DNA
Discussion
Can you tell
the story?
amino
acids
exon
intron
tRNA
pre-mRNA
5' GTP cap
mature mRNA
poly-A tail
large ribosomal subunit
polypeptide
5'
small ribosomal subunit
tRNA
E P A
ribosome
3'
Bacterial chromosome
Protein
Synthesis in
Prokaryotes
Psssst…
no nucleus!
Cell
membrane
Cell wall
Transcription
mRNA
Prokaryote vs. Eukaryote genes
 Prokaryotes
 Eukaryotes


DNA in cytoplasm
circular
chromosome
naked DNA

no introns





DNA in nucleus
linear
chromosomes
DNA wound on
histone proteins
introns vs. exons
introns
come out!
intron = noncoding (inbetween) sequence
eukaryotic
DNA
exon = coding (expressed) sequence
Translation in Prokaryotes
 Transcription & translation are simultaneous
in bacteria
DNA is in
cytoplasm
 no mRNA
editing
 ribosomes
read mRNA
as it is being
transcribed

Mutations
Mutations
 Mutations can change the protein
product, create more protein product,
and/or create less protein product
 They can be beneficial, detrimental, or
neutral
Depends on environment!
 Recall natural selection. Optimization,
benefit vs cost, in that place at that
time, etc.

Mutations
 Point mutations
single base change
 base-pair
substitution

 silent mutation
 no amino acid change
 redundancy in code
 missense
 change amino acid
 nonsense
 change to stop codon
When do mutations
affect the next
generation?
Point mutation leads to Sickle cell anemia
What kind of mutation?
Missense!
Sickle cell anemia
 Primarily African descent - recall malaria

Autosomal codominant/recessive
inheritance pattern
Mutations
 Frameshift

shift in the reading
frame
 changes everything
“downstream”

insertions
 adding base(s)

deletions
 losing base(s)
Where would this mutation
cause the most change:
beginning or end of gene?
Discussion: Which kind of mutation tends to have
the more profound effect?
THE RAT AND THE CAT ATE THE RED BAT
Deletion
THE RTA NDT HEC ATA TET HER EDB AT
Insertion
THE RAA TAN DTH ECA TAT ETH ERE DBA T
Point
THE RQT AND THE CAT ATE THE RED BAT
Cystic fibrosis
 Primarily whites of
European descent

strikes 1 in 2500 births
 1 in 25 whites is a carrier (Aa)
 Seems to be primarily due to a founder effect

normal allele codes for a membrane protein
that transports Cl- across cell membrane
 defective or absent channels limit transport of Cl- (& H2O)
across cell membrane
 thicker & stickier mucus coats around cells
 mucus build-up in the pancreas, lungs, digestive tract &
causes bacterial infections

without treatment children die before 5;
with treatment can live past their late 20s
Large-Scale
Mutations
Can be very
deleterious… or very
beneficial!
 Example: gene duplication, often
advantageous. Can:

 provide new phenotypes
 provide a “back-up” in case one of the
genes suffers a deleterious mutation
 allow one gene to maintain its original
function while another copy of the gene
evolves a different function
Discussion
 Cancer is caused by mutations to gene
sequences that control the pace of the
cell cycle.
 Scientists were originally baffled as to
how so many things - sunlight,
cigarette smoke, family history, viral
infection - could cause cancer.

How COULD so many different things
ALL be carcinogenic?
Causes
 Mutations can be caused by:



Errors in DNA Replication
Errors in DNA repair mechanisms
External factors that affect the chemical
structure of DNA
 Radiation, ionizing or ultraviolet
 Mutagenic chemicals
 Chemicals that react with DNA
 Base analogs, chemicals that can bond in place of a
nitrogenous base
 Ex: 5BU can bond in place of a T, and bonds with
A. But it periodically and spontaneously shifts
into an isomer that bonds with G instead, causing
a replication error.
Molecular Genetics + Inheritance
 Protein synthesis explains why inheritance
elements work the way they do…
 Think in terms of protein synthesis…
What could cause two alleles to be
codominant? (i.e. if you have both alleles, you
have both phenotypes simultaneously)
 Why might a heterozygote have a more
advantageous genotype than a homozygote?

Viruses
What is a virus?
 Genetic material (either DNA or RNA)
within a protein capsid (or envelope or
capsule)

An infectious agent, but NOT a
cell, and cannot reproduce
without enlisting a cell
Viral Reproduction
 DNA viruses use single- or double-stranded
DNA.
 RNA viruses include retroviruses, which have
RNA as their genetic material and later
convert it to DNA.
 Both classes have highly efficient means of
replicating that generate high genetic
diversity, allow for rapid evolution, rapid
acquisition of new phenotypes
Viral Reproduction
 Two cycles of
reproduction.
Both:




involve introduction of
viral genetic material
into host cell
use the host cell’s
genetic/protein
synthesis machinery
allow for mutations to
occur in both viral and
host DNA through the
usual mechanisms
can transfer DNA
between viruses, if the
host has multiple
infections
Lytic Cycle
 One virus reproduces
many progeny viruses




Virus attaches to,
penetrates cell
Releases its genetic
material into cell
Viral DNA separate from
host DNA
DNA polymerase
transcribes viral DNA
 ->Virus is now directing
some of the cell’s
ribosomal activity
Lytic Cycle
 Viral genes encode
viral enzymes and
capsid proteins

The translation
products are new
viruses!
 Many viral progeny
are produced, the
host cell bursts
(“lyses”), progeny
are released
Lysogenic Cycle
 Some viruses can instead
integrate their DNA into the host
cell’s, establishing a dormant
(“latent”) infection

Viral DNA copied and
transmitted like the host’s own
DNA
 Sleeper agents! :O

But the host cell survives this
cycle, and can even acquire new
properties
 Ex: some bacteria are more
pathogenic when carrying viral
DNA
Discussion
 From the virus’s perspective, what are
the advantages and disadvantages of
being in the lytic cycle? The lysogenic
cycle?
Retroviruses
 Retroviruses carry RNA
RNA transcriptase converts RNA to
DNA once “injected” into the host
 RNA -> DNA -> RNA -> Protein

Ex:
leukemiaviruses,
HIV, hepatitis B
viruses
Consequence of retrovirulence
 RNA viruses lack replication errorchecking mechanisms
=Higher rate of mutation
=Rapid evolution
 Discussion: How does this feature
contribute to the pathogenicity of
retroviruses such as HIV?
Viral Integration into
Host Genome
 Some lysogenic infections never go
away……… EVER
8% of the human genome is viral! More
than 100,000 gene regions
 From the perspective of viral DNA,
permanent non-damaging incorporation
into a host genome = highly effective
means of reproduction!

 Not necessarily a very successful virus, but
a very successful gene!