DNA

DNA

Gregor Mendel – 1840’s

Conclusion:Heredity material was packaged in discrete transferable units; came up with law of segregation and law of independent assortment.

Thomas Morgan – early 1900’s

Discovered that fruit flies’ genes were associated with chromosome inheritance.

Conclusion:Chromosomes were known to be composed of proteins and DNA, so genes must be one of these two macromolecules

ExperimentIf dead (heat-killed) pathogenic bacteria was mixed in a culture with living harmless bacteria, the harmless bacteria would become deadly.

ConclusionTransformation in bacteria allows a change in genotype and phenotype due to the assimilation of external DNA by the cell.

FREDRICK GRIFFITH -1928

He separated the components of the heat-killed, deadly bacteria & divided it into smaller samples (proteins, lipids, carbohydrates, or nucleic acids) & left the other molecules intact. He then mixed each sample of the treated lethal strain with living samples of the non-lethal strain.

AVERY’S EXPERIMENT -1944

Only the DNA extract from the deadly bacteria would allow the live harmless bacteria to become deadly.

AVERY’S CONCLUSION

HERSHEY AND CHASE - 1952

Conclusion: The DNA molecule entered the bacteria cell & not the protein. This showed that DNA and not protein controls traits that are passed on.

CHARGAFF - 1947Experiment:

Studied the composition of DNA and the concentration of each of the nitrogenous bases.

Conclusion:DNA base composition varies from one species to another; bases are not present in equal amounts in any one species but they are found in a predictable ratio; concentration of T=A and concentration of C=G.

Franklin and Wilkins

Experiment: Took x-ray diffraction pictures of DNA in its different forms

Conclusion:Discovered that the B form of DNA was double helix in structure.

Watson and Crick

Experiment: Built a 3-D model that reflected the base pairing rules determined by Chargraff & the distance between bases suggested by Franklin’s X-ray photos.

Conclusion:DNA was a double helix that made one full turn every 3.4nm with bases 0.34nm apart & sugar/phosphate molecules on the outside of the ladder.

So what are the various parts of DNA?

• Nitrogen bases– Adenine– Cytosine– Guanine– Thymine

• Phosphates• Deoxyribose• Hydrogen bonds

DNA Molecule

Sugar and phosphate backbones are antiparallel (their subunits run in opposite directions)

Adenine and guanine are purines (both have 2 organic rings)Cytosine and thymine are pyrimidines (both have 1 organic ring)

Adenine forms 2 hydrogen bonds with thymineCytosine forms 3 hydrogen bonds with guanine

WHY 5’ AND 3’?

DNA Replication

What is the purpose of DNA Replication?

• To produce a copy of DNA identical to the original in preparation for mitosis or meiosis.

Meselson and Stahl experiment

Conclusion: DNA replication follows the semiconservative model.

1st replication in the 14 N medium produced a band of hybrid. This eliminated the conservative model.

2nd replication produced both light and hybrid DNA this eliminated the dispersive model & supported the semiconservative model.

E. coli vs Human DNA Replication

E. coli • Has a single chromosome• 4.6 million nucleotide pairs• Can replicate its

chromosome in less than an hour.

Human• 46 DNA molecules; each in

a chromosome• 6 billion nucleotide pairs• Can replicate all

chromosomes in a few hours

Replication process is similar in prokaryotes and eukaryotes.

Some of the “players” involved…• DNA Polymerase- adds nucleotides to a preexisting chain.

• Ligase- joins the sugar phosphate backbones of all Okazaki fragments.

• Primase- synthesizes the primer that’s 5-10 nucleotides long.

• Helicase- unzips the DNA

• Topoisomerase-relieves the strain of overtwisting DNA by braking, swiveling, & rejoining DNA strands.

DNA unwinds & unzips with the help of helicase

In order for replication to begin a primer is

needed & it is an RNA primer

The primer is about 5-10 nucleotides long

The new DNA strand starts from the 3’ end of the RNA primer

DNA Polymerase adds nucleotides to the preexisting strand

Replication occurs from 5’ to 3’

In eukaryotes Okazaki fragments are 100-200 nucleotides long.

Leading strand- is made going towards the replication fork and is continuous

Lagging strand- is made going away from the replication fork and is synthesized discontinuously, as a series of segments called Okazaki fragments.

Leading strand needs only one primer & lagging strand needs a primer for each Okazaki fragment.

Ligase- joins sugar-phosphate backbone.

DNA polymerase I – removes the primer and replaces it with DNA nucleotides; one by one

DNA polymerase III-Continuously synthesizes the leading strand

How is replication of one side of each double strand different than the other?

• Because bases can only be added in the 5’ to 3’ direction, the 3’ to 5’ strand must be assembled in fragments that are later annealed together by a ligase protein.

Proofreading and Repairing

DNA

Errors amount to 1 in 10 billion nucleotides in the final DNA product

Initial pairing error amount to 1 in 100,000 more common.

How is the new strand ensured to be identical?

• The bases are matched in a consistent pattern• The daughter strands are half new, half old• There is a proofreading mechanism that

checks for errors in both strands.

Why are mistakes made?

• Spontaneous chemical changes under normal conditions.

• Exposure to mutagens– EX: cigarette smoke and X-rays

There are many different DNA repair enzymes.-E. coli has 100 known repair enzymes-Humans have 130 identified repair enzymes

Teams of enzymes detect & repair damaged DNA, such as this thymine dimer (often caused by UV radiation), which distorts the DNA molecule.

A nuclease enzyme cuts the damaged DNA strand at 2 points, & the damaged section is removed.

Repair synthesis by a DNA polymerase fills in the missing nucleotides.

DNA ligase seals the free end of the new DNA to the old DNA, making the strand complete.

If your body is unable to repair the thymine dimer…

Replicating the ends of DNA moleculesThis kind of thing does not occur prokaryotes with a

circular chromosome

The primer on the end is removed but can’t be replaced

with DNA because DNA polymerase can only add

nucleotides to the 3’ end of a preexisting polynucleotide

Because of the 5’ to 3’ constraint & the need for each replication event to by initiated with a primer, one side of the replication will be inhibited at the

end. What will happen to the length of the chromosome after numerous replications?

It will get shorter

What is done to compensate for this problem?

Telomeres (located at the ends of DNA molecules) are made of repeated units that are non-coding so that, as they get shorter, no genes are lost.

The enzyme telomerase lengthens telomeres in germ cells

Cells can only go through a limited number of replications before they are put to death.

plus

also

Let’s make some DNA

• Red = phosphate• White = deoxyribose• Yellow = adenine• Blue = thymine• Orange = cytosine• Green = guanine• Pink = uracil• Plastic connectors = hydrogen bonds

Let’s Replicate!!!with narrative

How Does a Gene Become a Protein?

With a lot of help, I’ll tell you that!!!

Let’s start with the 2 nucleic acids involved

DNA and RNAThese molecules have structural similarities and differences that

define function.

Compare DNA to RNA

• Made of nucleotides• Connected by covalent

bonds to form a linear molecule from 5’ to 3’

• Contains deoxyribose• Nitrogen bases A,T,G, & C• Double stranded• Restricted to the nucleus

(eukaryotes)

• Made of nucleotides• Connected by covalent

bonds to form a linear molecule from 5’ to 3’

• Contains ribose• Nitrogen bases A,U,C, & G• Single stranded• Able to travel out of the

nucleus (eukaryotes)

Comparison of DNA to RNA

DNA RNA

Is Uracil a purine or a pyrimidine?• Since thymine is a pyrimidine and in essence

uracil replaces thymine in RNA it would make sence that uracil is also a pyrimidine.

Wouldn’t it?

The sequence of the RNA bases, together with the structure of the RNA molecule, determines RNA

function.

• mRNA: carries information from the DNA to the ribosome.

• tRNA: are molecules that bind specific amino acids and allow information in the mRNA to be translated to a linear peptide sequence.

• rRNA: are molecules that are functional building blocks of ribosomes.

• RNAi: plays a role in regulation of gene expression at the level of mRNA transcription.

• To be discussed later

Genetic information flows from a sequence of nucleotides in a gene to a sequence of amino acids in a protein

This occurs in two parts

Transcription• Is the synthesis of RNA using one

side of a segment of a DNA strand.– The DNA segment serves as a

template.• The RNA made is mRNA.

– It is antiparallel to the DNA template– mRNA is made from 5’ to 3’ (reading

the DNA in a 3’ to 5’ direction).• This is all completed in the nucleus.

• RNA Polymerase opens the DNA strands & joins the RNA nucleotides that are complimentary to the DNA template.– Needs no primer to begin

• Bacteria have a single type of RNA polymerase that synthesizes all types of RNA.

• Eukaryotes have at least 3 types of RNA polymerase.

An example of a promoter is the TATA

box

Termination of Transcription

• Differs between bacteria and eukaryotes– In bacteria

• Go through a terminator sequence in DNA.• Once RNA polymerase hits the terminator signal it releases

from the DNA and the mRNA that was being made.

– In eukaryotes• RNA Polymerase II transcribes a sequence on the DNA

which codes for a polyadenylation signal (AAUAAA) in the pre-mRNA.

• About 10-35 nucleotides later proteins cut the pre-mRNA from the polymerase & undergoes processing…

Modifying of the pre-mRNA in Eukaryotes

• Both ends of the mRNA transcript are altered.

• In most cases, certain interior sections are cut out and the remaining pieces are spliced together.

• These actions produce a mRNA molecule that is ready for action!!!

RNA Processing

The cap and tail:-help facilitate the mRNA leaving the nucleus & help protect the mRNA strand from degradation by hydrolytic enzymes.- help the ribosomes attach to the 5’ end of the mRNA

For ribosome binding

More RNA Processing

• RNA splicing:– Removing portions of the RNA molecule & putting

the other ends together.– The parts to be “edited out” are interspersed

between the coding segments.• The segments that intervene with the coding segments

are called: introns• The segments that will eventually be expressed & exit

the nucleus are called: exons

Average length of transcription unit = 8000 nucleotides, but average size protein is 400 amino acids so only about 1,200 nucleotides long. This indicates long noncoding stretches of nucleotides which happen to be interspersed in the coding segments.

HOW IS PRE-mRNA SPLICING CARRIED OUT?

A small nuclear ribonucleoproteins (snRNPs) recognize the splice sites.

These are located at the end of introns.

Composed of RNA & protein

Several snRNPs and additional proteins form a larger assembly: spliceosome

These molecules release introns & join the exons

Why have introns?

• One idea is that introns play regulatory roles in the cell.– Splicing process is necessary for mRNA to leave

the nucleus.• Consequence for having introns & exons

– Genes are known to give rise to 2 or more different polypeptides, depending on which segments are treated as exons during RNA processing = alternative RNA splicing.

Translation

Divided into 3 stages: initiation, elongation, & termination

Translation• Molecular components of translation

– mRNA: has nucleotide triplets called: codons• Written in the 5’ to 3’ direction

– tRNA: has nucleotide triplets called: anticodons• They are complimentary to the codons• Main function is to transport amino acids to the

ribosome & drop off the amino acid to add to the polypeptide chain.

– Ribosomes: made of rRNA and proteins• Adds each amino acid brought by the tRNA to the

growing end of the polypeptide chain.

Stage 1: Initiation

• mRNA interacts with the rRNA of the ribosome to initiate translation at the (start) codon & travels from the 5’ to 3’ end.

• The sequence of nucleotides on the mRNA is read in triplets called codons.

• Each codon encodes a specific amino acid, which can be deduced by using a genetic code chart. Many amino acids have more than one codon.

• tRNA brings the correct amino acid to the correct place on the mRNA.

• The amino acid is transferred to the growing peptide chain, with the help of ATP.

The process continues along the mRNA until a “stop” codon is reached

Stage 2 : Elongation

Stage 3: Termination

• A release factor (protein) binds directly to the stop codon.– This causes an addition of a water molecule

instead of an amino acid to the polypeptide chain.• This breaks the bond between the chain & the tRNA.

• The process terminates by the ribosome falling off the mRNA strand releasing the newly synthesized peptide chain.

The polypeptide chain folds and becomes active

What happens to the mRNA strand?

They eventually degrade in the cytoplasm and become free floating

nucleotides once again.

Transcription and translation can happen simultaneously.

In other words, translation of an mRNA molecule begins while still being transcribed.

Picture summary of transcription and translation in a eukaryotic cell.

Each amino acid attaches to its proper tRNA with the help of a specific enzyme & ATP.

Questions Transcription Translation

Where? Nucleus Cytosol/Cytoplasm

What is used as a template?

DNA mRNA

What is used to synthesize the new

strand?

RNA Polymerase Ribosomes

What is the new strand made of?

RNA Amino acids

LET’S MAKE SOME PROTEIN!I need 4 volunteers

Which parts played what in transcription & translation?

Master Chef = RNA Polymerase

Prep-chef= ribosomeDNA

Scrap paper= mRNA

Ingredients= amino acids

Customer 1= receptor mediated cell signal

Customer 2= cell signal- hormone

Tabs on cookbook= TATA boxes (telling chef where to find the recipe/gene

Now it’s your turn

• You will get together with 4-5 people in class.

• You will now create a completely different analogy for the transcription & translation process.

• You have 20 minutes to come up with your analogy & then we will present them

How do you know the code from the mRNA to the amino acid?

How Genes Influence Traits

Genes specify the amino acid sequence of proteins The amino acid sequence determines the

shape and activity of proteinsProteins determine a majority of what the

body looks like and how it functions

Fig. 8.11 The journey from DNA to phenotype

Fig. 8.11 The journey from DNA to phenotype

HIV - HIV is a unique virus in that its genetic material is a single-stranded RNA. The flow of genetic information travels from RNA to DNA.

Once the HIV virus enters the white blood cells, it activates an enzyme called reverse transcriptase.

This enzyme uses the RNA of the virus to synthesize complimentary double stranded DNA. The process of reverse transcription is very error-prone, hence there is a large degree of mutation which is why finding a cure for AIDS is so difficult.

This new DNA integrates itself into the host genome & becomes transcribed and translated for the assembly of new viral progeny.

RETROVIRUS

Targeting polypeptides to specific locations

• Two populations of ribosomes–Free and bound

• Free are in the cytosol– Make proteins that stay and function here

• Bound ribosomes– Usually attached to the cytosolic side of the

endoplasmic reticulum (ER) or the nuclear envelope.

– Make proteins of the endomembrane system as well as proteins secreted from the cell (ex: insulin)

One gene/one polypeptide hypothesis

• In the 1940’s experimental work led to the hypothesis:

• Every one gene of DNA produce one enzyme

• This was amended to include all proteins.• It was later discovered that many proteins are

actually composed of more than one polypeptide.

• This led to the proposal that each individual polypeptide required one gene.

In the last few years…• Researchers have discovered that at least

some genes aren’t quite that straightforward.

– For Example:• One gene may lead to a single mRNA molecule, but the

mRNA molecule may then be modified in many different ways.

• Each modification may result in a different polypeptide.

So…what is a gene?

A region of DNA that can be expressed to produce a final functional product that is either a polypeptide or an RNA molecule.

Point Mutations

What can affect protein structure

and function?

Types of Small-Scale Mutations

• Substitutions:– Nucleotide-pair substitution

• Replacement of one nucleotide & its partner with another pair of nucleotides.

– Results in one of the following: silent mutation, missense mutation, or nonsense mutation.

• Insertions & deletions– Additions or loses of nucleotide pairs in a gene

• Disastrous effect on the resulting protein• Whenever the number of insertions or deletions aren’t a

multiple of three = frameshift mutation.

EX: Point mutation: Sickle-cell disease