DNA The Genetic Material

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!