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DNA The Genetic Material. 1947. 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%. Rules A = T C = G. - PowerPoint PPT Presentation
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DNAThe 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%
1947
That’s interesting!What do you notice?
RulesA = TC = G
Structure of DNA Watson & Crick
developed double helix model of DNA other leading scientists working on question:
Rosalind FranklinMaurice WilkinsLinus Pauling
1953 | 1962
Franklin Wilkins Pauling
Watson and Crick1953 article in Nature
CrickWatson
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
number the carbons! Think Five =
Phosphate
OH
CH2
O
4
5
3 2
1
PO4
N base
ribose
nucleotide
This will beIMPORTANT!!
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
3
5
5
3
Bonding in DNA
….strong or weak bonds?How do the bonds fit the mechanism for copying DNA?
3
5 3
5
covalentphosphodiester
bonds
hydrogenbonds
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!
2007-2008
DNA Replication
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 Large team of enzymes coordinates replication
Let’s meetthe team…
Replication: 1st step Unwind DNA
helicase enzyme unwinds part of DNA helix (hence “helicase,”
AMAZING I KNOW) stabilized by single-stranded binding proteins
single-stranded binding proteins replication fork
helicase
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
DNAPolymerase III
Replication: 2nd step
But…We’re missing something!
What?
But where’s theENERGY
for the bonding!
Build daughter DNA strand add new
complementary bases Polymerization, an
anabolic process DNA polymerase III
energy
ATPGTPTTPCTP
Energy of ReplicationWhere does energy for bonding usually come from?
ADPAMPGMPTMPCMPmodified nucleotide
energy
We comewith our ownenergy!
And weleave behind anucleotide!
YourememberATP!
Are there other ways
to get energyout of it?
Are thereother energynucleotides?You bet!
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
Adding bases can only add
nucleotides to 3 end of a growing DNA strand need a “starter”
nucleotide to bond to
strand only grows 53
DNAPolymerase III
DNAPolymerase III
DNAPolymerase III
DNAPolymerase III
energy
energy
energy
Replication energy
3
3
5B.Y.O. ENERGY!The energy rules
the process
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?
energy
35
5
5
3
need “primer” bases to add on to
energy
energy
energy
3
no energy to bond
energy
energy
energy
ligase
3 5
Limits of DNA polymerase III can only build onto 3 end of
an existing DNA strand
Leading & Lagging strands
5
5
5
5
3
3
3
53
53 3
Leading strand
Lagging strand
Okazaki fragments
ligase
Okazaki
Leading strand continuous synthesis
Lagging strand Okazaki fragments
Short DNA fragments
joined by ligase “spot welder” enzyme
DNA polymerase III
3
5
growing replication fork
DNA polymerase III
Replication fork / Replication bubble
5
3 5
3
leading strand
lagging strand
leading strand
lagging strandleading strand
5
3
3
5
5
3
5
3
5
3 5
3
growing replication fork
growing replication fork
5
5
5
5
53
3
5
5lagging strand
5 3
DNA polymerase III
RNA primer built by primase, serves as starter
sequence for DNA polymerase III @ start of leading strand, and at start of
each Okazaki fragment
But there’s yet another problem! can only build onto 3 end of
an existing strand
Starting DNA synthesis: RNA primers
5
5
5
3
3
3
5
3 53 5 3
growing replication fork
primase
RNA
DNA polymerase I removes sections of RNA
primer and replaces with DNA nucleotides
But DNA polymerase I still can only build onto 3 end of an existing DNA strand. One primer can’t be acted upon…
Replacing RNA primers with DNA
5
5
5
5
3
3
3
3
growing replication fork
DNA polymerase I
RNA
ligase
Ligase Connects
strands
Loss of bases at 5 ends in every replication chromosomes get shorter with each replication limit to number of cell divisions
DNA polymerase III
Chromosome erosion
5
5
5
5
3
3
3
3
growing replication fork
DNA polymerase I
RNA
Houston, we have a problem!
Repeating, non-coding sequences at the end of chromosomes = protective cap to erode instead of gene sequence
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
Telomeres
5
5
5
5
3
3
3
3
growing replication fork
TTAAGGGTTAAGGGTTAAGGG
Replication fork
3’
5’
3’
5’
5’
3’
3’ 5’
helicase
direction of replication
SSB = single-stranded binding proteins
primase
DNA polymerase III
DNA polymerase III
DNA polymerase I
ligase
Okazaki fragments
leading strand
lagging strand
SSB
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?
proteins cells bodies
How does DNA code for cells & bodies? how are cells and bodies made from the
instructions in DNA
DNA
The “Central Dogma” Flow of genetic information in a cell
How do we move information from DNA to proteins?
transcriptiontranslation
replication
proteinRNADNA trait
DNA gets all the glory, but proteins do all the work!
Transcription
fromDNA nucleic acid language
toRNA nucleic acid language
RNA as opposed to DNA ribose sugar N-bases
uracil instead of thymine U : A
single stranded lots of RNAs
mRNA, tRNA, rRNA…
RNADNAtranscription
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
RNA polymerase or RNAP
template strand
rewinding
mRNA RNA polymerase
unwinding
coding strand
DNAC C
C
C
C
C
C
C
C CC
G
GG
G
G G
G G
G
G
GAA
AA A
A
A
A
A
A A
A
AT
T T
T
T
T
T
T
T T
T
T
U U
5
35
3
3
5build RNA 53
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
Matching bases of DNA & RNA Match RNA bases to DNA
bases on one of the DNA strands
U
A G GGGGGT T A C A C T T T T TC C C CA A
U
UU
U
U
G
G
A
A
A C CRNA
polymerase
C
C
C
C
C
G
G
G
G
A
A
A
AA
5' 3'
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
Eukaryotic genes have junk!
Eukaryotic genes are not continuous exons = the “real gene”
expressed / coding introns = the “junk”
inbetween sequence
exon = coding (expressed) sequence
intron = noncoding (inbetween) sequence
intronscome out!
mRNA splicing
eukaryotic DNA
exon = coding (expressed) sequence
intron = noncoding (inbetween) sequence
primary mRNAtranscript
mature mRNAtranscript
pre-mRNA
spliced mRNA
Post-transcriptional processing eukaryotic mRNA needs work after transcription primary transcript = pre-mRNA mRNA splicing
edit out introns make mature mRNA transcript
~10,000 bases
~1,000 bases
RNA splicing enzymes
snRNPs
exonexon intron
snRNA
5' 3'
spliceosome
exonexcisedintron
5'
5'
3'
3'
3'
lariat
exonmature mRNA
5'
No, not smurfs!“snurps”
snRNPs small nuclear RNA proteins
Spliceosome several snRNPs recognize splice
site sequence cut & paste gene
mRNA splicing
eukaryotic DNA
exon = coding (expressed) sequence
intron = noncoding (inbetween) sequence
primary mRNAtranscript
mature mRNAtranscript
pre-mRNA
spliced mRNA
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.
~10,000 bases
~1,000 bases
Alternative splicing Alternative mRNAs produced from same gene
different segments treated as exons one gene can thus make multiple proteins
Starting to gethard 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?
A A AA
A3' poly-A tail
mRNA
5'5' cap
3'
G PPP
50-250 A’s
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
Translation
fromnucleic acid language
toamino acid language
How does mRNA code for proteins?
TACGCACATTTACGTACGCGGDNA
AUGCGUGUAAAUGCAUGCGCCmRNA
Met Arg Val Asn Ala Cys Alaprotein
?
How can you code for 20 amino acids with only 4 nucleotide bases (A,U,G,C)?
4
4
20
ATCG
AUCG
AUGCGUGUAAAUGCAUGCGCCmRNA
mRNA codes for proteins in triplets
TACGCACATTTACGTACGCGGDNA
AUGCGUGUAAAUGCAUGCGCCmRNA
Met Arg Val Asn Ala Cys Alaprotein
?
codon
Cracking the code Francis Crick
determined 3-letter (triplet) codon system Codon = 3 mRNA bases that will match to 1 amino acid
WHYDIDTHEREDBATEATTHEFATRATWHYDIDTHEREDBATEATTHEFATRAT 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”
Start codon AUG methionine
Stop codons UGA, UAA, UAG
Why is thewobble good?
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?
TACGCACATTTACGTACGCGGDNA
AUGCGUGUAAAUGCAUGCGCCmRNA
aminoacid
tRNA anti-codon
codon
5 3
3 5
3 5
UAC
MetGCA
ArgCAU
Val
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
Met
5'
3'
UUA C
A G
APE
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
E site (exit site) empty tRNA
leaves ribosome from exit site
Building a polypeptide Initiation
brings together mRNA, ribosome subunits, initiator tRNA
Elongation adding amino acids based on
codon sequence
Termination end codon 123
Leu
Leu Leu Leu
tRNA
Met MetMet Met
PE AmRNA5' 5' 5' 5'
3' 3' 3'3'
U UA AAACC
CAU UG G
GUU
A AAAC
CC
AU UG GGU
UA
AAAC
CC
AU UG GGU U
A AACCA U UG G
G AC
ValSer
AlaTrp
releasefactor
AA A
CCU UGG 3'
http://www-class.unl.edu/biochem/gp2/m_biology/animation/gene/gene_a3.htmlhttp://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
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’
Can you tell the story?
DNA
pre-mRNA
ribosome
tRNA
aminoacids
polypeptide
mature mRNA
5' GTP cap
poly-A tail
large ribosomal subunit
small ribosomal subunit E P A
5'
3'
RNA polymerase
exon intron
tRNA
Discussion
Protein Synthesis in Prokaryotes
Bacterial chromosome
mRNA
Cell wall
Cellmembrane
Transcription
Psssst…no nucleus!
Prokaryote vs. Eukaryote genes Prokaryotes
DNA in cytoplasm circular
chromosome naked DNA
no introns
Eukaryotes DNA in nucleus linear
chromosomes DNA wound on
histone proteins introns vs. exons
eukaryoticDNA
exon = coding (expressed) sequence
intron = noncoding (inbetween) sequence
intronscome out!
Transcription & translation are simultaneous in bacteria DNA is in
cytoplasm no mRNA
editing ribosomes
read mRNA as it is being transcribed
Translation in Prokaryotes
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.
When do mutationsaffect the nextgeneration?
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
Point mutation leads to Sickle cell anemiaWhat 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?
THE RAA TAN DTH ECA TAT ETH ERE DBA T
Discussion: Which kind of mutation tends to have the more profound effect?
THE RAT AND THE CAT ATE THE RED BAT
THE RTA NDT HEC ATA TET HER EDB ATDeletion
Insertion
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 error-
checking 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!