molecular bio

Molecular Biology

the study of gene structure and function at the molecular level

Gene

sequence of DNA that codes for a protein

Central Dogma

Gene expression occurs in two major steps - transcription and translation.

Gene Expression Steps

Genes (sequences of DNA), are inherited. DNA is replicated during cell division so that each somatic cell of an organism contains the same DNA (gametes, or reproductive cells undergo meiosis, and so they contain less DNA). DNA undergoes the first step, transcription, to make mRNA. Translation is the second step, and this is used to make protein.

How did we learn that DNA exists?

Pre-historic people recognized the existence of heredity

Domestication of animals (8000 - 1000 BC)

Cultivation of plants (5000 BC)

Desirable and undesirable varieties/traits of animals and plants

could be selected

Particulate Theory

Aristotle felt that parts, or particles were passed from each parent to an offspring

Gregor Mendel

Studied how traits were inherited in pea plants units of inheritance were passed from parents to offspring

Factors

were responsible for inheritance

some unit of inheritance was being passed from parent to offspring

Next major advancement: inherited factors are located on chromosomes

it was known that gametes (reproductive cells) are haploid - the number of chromosomes in these cells are cut in half

Thomas Hunt Morgan

credited with establishing that chromosomes are responsible for inheritance through the use of his fruit fly experiments

He mated white-eyed males and red-eyed females.

Red eyes are wild-type. In the first generation from this cross, all flies are red-eyed, which is not surprising because red eyes are dominant.

But in the F2 generation, white eyes appeared only in the males.

Linked Inheritance

Genes that are located on the same chromosome are "linked" and are usually inherited together

Crossing Over

chromosomes can exchange genetic information during prophase I of meiosis - mixing up the traits that can be inherited

Traits that are inherited more frequently together are closer together on the chromosome

Friedrich Miescher

discovered the existence of DNA

"Nuclein" precipitated in acid, and could dissolve again in alkaline conditions (not like protein) - so he knew that this was not protein, but had to be a different type of molecule

Genetic Material

Must provide information responsible for traits

Must provide information for replication of an organism

Must itself be replicated

Griffith

He was studying the bacteria that causes pneumonia. One strain, S strain, had a smooth coat, and could therefore evade the immune system and was pathogenic in mice. The R strain had a rough coat, and does not cause disease.

Griffith Mouse Experiment

Mice treated with S: S causes disease, the mouse will die

Mice treated with R: R does not cause disease, mouse will live

Mice treated with heat-killed S: Heat will kill the bacteria - mouse will live

Mice treated with heat-killed S and live R: mouse dies.... Upon autopsy, both S and R living bacteria are found.... This could not be explained at the time it was done... it was explained years later

Avery

Explain Griffith

Isolated DNA from the S strain and added it directly to the R strain: the R strain was then "transformed" to become an S strain

Hershey and Chase

On the left, the phage protein was labeled with S-35. The protein coat is shown labeled in blue as a result. The phage are allowed to infect, and then they used a blender to mix up and separate the phage from the infected bacteria, which could be centrifuged.

On the right, the DNA was labeled with P32. The labeled phages were allowed to infect, and the parent phages were again removed by blending and the infected bacteria were centrifuged.

They knew that the bacteria pelled were infected, and therefore would have offspring phages being made inside - what was responsible for the inherited traits of these phages? The could only detect P32 inside the infected bacteria, so DNA was responsible for inheritance of the phages.

Garrod

"inborn error of metabolism"

alkaptonuria

It was known that this was inherited as a recessive trait, and Garrod realized that there was one defective gene that caused this

So one defective gene resulted in one defective enzyme

Beadle and Tatum

one gene one enzyme

studying the growth of the mold Neurospora

Chargaff

first to realize that the nitrogenous bases of DNA were not found in equal number (A, G, C, T)

The #A's were equal to the #T's, and the #Cs were equal to the #Gs

He did not know about complementary base pairing, but he knew that the ratios existed

Franklin and Wilkins

X-ray diffraction studies of DNA - isolating DNA, hitting it with an X-ray to see the pattern of its structure

Watson and Crick

Could figure out that DNA had a helical structure.

B Form of DNA

proposed by Watson and Crick

high humidity (92%)

Base pairs are horizontal

right-handed - the highest phosphate-sugar chain begins on the right and wraps around to the left

A-DNA

right-handed

found at 75% relative humidity

Plane of the base pairs tilts 20 degrees from horizontal

11 bp per turn instead of 10 bp per turn

each turn occurs in 31A instead of 34A

found in hybrids of DNA and RNA, dsRNA

Z-DNA

alternating purines and pyrimidines

least compact; function not known

left-handed

Linear DNA

DNA is usually found as a closed structure (lacking free ends) - even if it is linear

linear DNA is held looped out on a protein scaffold, but it is held in the scaffold - keeping it a closed structure

Circular DNA

found in bacterial and viral genomes and in eukaryotic cells in large loops

Supercoils

introduced into DNA when it is twisted around its own axis, can only happen in closed structures

Molecules that are linear or that lack supercoiling: relaxed

Separating Double Helix

harder to separate G's and C's

The easiest way to accomplish this is by using heat since heat can break hydrogen bonds. Denaturing DNA is also called "melting" DNA

Tm

the temperature at which half of the DNA strand is melted

This is greatly influenced by GC content

DNA Light Absorbance

How much DNA is melted can be determined by measuring absorbance at 260 nm. DNA absorbs light at 260 nm, and does this more efficiently when it is single-stranded. In this graph, you can see that as the temperature increases, the absorbance at 260 nm goes up

Other Factors that Influence Tm

Organic solvents:

High pH:

Lowering the salt concentration:

Organic SOlvents

functional groups can form H-bonds with the nitrogenous bases, preventing them from hydrogen bonding with each other

High PH

hydroxide ions (OH-) can form bonds with the hydrogens involved in hydrogen bonding between nitrogenous groups, pulling them apart

Lowering the Salt Concentration

this can remove ions that block the negative charges on the DNA backbone, which causes the two strands to then repel each other

Annealing or Renaturation

heat it at 95 degrees and then just turn off the heat block and leave it overnight - it will gradually cool and renature

Factors that influence renaturation efficiency

Temperature

DNA Concentration

Renaturation Time

Temperature

best 25°C below the Tm

high enough = rapid diffusion of DNA

low enough = doesn't promote denaturation

DNA Concentration

higher the concentration = more likely to come in contact

Renaturation Time

longer time allowed for annealing = more annealing will occur

CoT Curve

Describe the renaturation of DNA

reassociation depends on time and concentration

CoT1/2

time and concentration it takes for ½ the DNA to be renatured

Falling CoT Curve

represent re-associated DNAs.

At the top of the curve, 0% is renatured, when the bottom is reached, 100% is renatured.

Complexity of DNA

DNA influences the concentration/time (Cot) needed for reassociation

The complexity is the amount of unrepetitive sequence in the DNA molecule

Highly Repetitive

short sequences of DNA

satellite DNA: found at centromeres

telomere sequences

(10% of mouse genome)

Moderately Repetitive

genes for ribosomal RNA

(30% of mouse genome)

Single-Copy

not repetitive

protein-encoding regions

Hybridization

Putting together two strands of different origin

(PCR, Northern/Southern blotting)

Same considerations as reassociation:

PCR

You need to anneal primers to template DNA

the primers need to be rich in GC content (especially at the 3' end, as this is the end that needs to be extended with new nucleotides during PCR)

The concentration of primers in the reaction needs to be high enough as well

the Tm of each member of the pair of primers should be similar, and the annealing step of the PCR reaction should be done at 25 degrees lower than the Tm of the primer

Southern Blots

blots detect the presence of a DNA sequence by using a tagged ssDNA probe to bind to it

Northern Blots

detecting the presence of an RNA sequence by using a tagged ssDNA probe to bind to it

Blotting Considerationas

Concentration of probe needs to be high enough

Tm has to be considered when planning the experimental conditions: cannot incubate the blot too hot, or the probe will not bind to the sequence

Low Stringency

this allows maximum amount of probe to bind to sequences on the membrane

Base mismatches are more tolerated.

Typically it is done around 55 degrees - hot enough to allow movement, but low enough that a lot of probe can bind.

It is usually an overnight incubation (long time)

Washes

used to increase the stringency- making sure there is more specificity between the probe and the target

Unbound probe, and probe that is poorly matched to sequences on the membrane would be removed;

the washes gradually lower the salt and higher the heat - making it harder for non-specific interactions between the probe and target to be maintained

C-Value

DNA content per haploid cell

Measured in molecular weight, bp, and length

Histones

proteins with highly positively-charged regions

are 5 types of histones (H2A, H2B, H3, and H4 make up the nucleosome bead, histone H1 attaches next to the bead structure

histones appear as beads on a string.

DNA wraps around the nucleosome (146 bp of DNA fit), and then the H1 histone is added at the end

Additional Packing

This beaded string structure then undergoes additional levels of packaging.

It will fold to form 30 nm-chromatin fibers, which then fold more to form looped domains

Looped DOmains

fold to form condensed chromosomes, the folding makes the chromosomes visible during mitosis

DNA Replication

Replication is linked to the cell cycle:

1). Initiation commits the cell to further division

2). Division of the cell can not occur until DNA replication

is finished

Meselson and Stahl

Proved that DNA replicates in a semiconservative fashion, confirming Watson and Crick's hypothesis. Cultured bacteria in a medium containing heavy nitrogen (15N) and then a medium containing light nitrogen (14N); after extracting the DNA, they demonstrated that the replicated DNA consisted of one heavy strand and one light strand

DNA Replication is Semidiscontinuous

DNA polymerase can only add nucleotides in a 5' to 3' direction... it needs a 3' end to attach the next nucleotide in the chain

However, when the fork opens on the right, the newly replicated piece at the top (shown in green) has a 5' end in the open region... DNA cannot be added to the 5' end... as the DNA unwinds further, the replication must be done backwards on this top strand, creating fragments (called Okazaki fragments) that are then linked together

DNA Ligase

an enzyme that eventually joins the sugar-phosphate backbones of the Okazaki fragments

DNA Replication Requires a Primer

DNA polymerase requires an existing 3' end to add nucleotides

Primer

short sequence of RNA (10- 12 bp) complementary to the DNA being replicated that is laid down first by a special type of RNA polymerase given the name "primase"

Replicon

Unit of DNA that is replicated from one origin of replication

Prokaryotic Genomes

single replicon

Plasmids

Extrachromosomal circular DNA

replicate autonomously

Eukaryotic Genomes

large number of replicons

each replicon is activated during S phase

Unidirectional

one replication fork

E. coli plasmid ColE1

Bidirectional

movement in two directions away from the origin

Some circular replicons, like the E. coli genome, are given the name "theta replication" because they look like the Greekletter theta, or an "eye" - this is bidirectional replication of a circular replicon

Eukaryotic Chromosomes

Replicate bidirectionally during S phase; replication continues along the replicon until it fuses with newly replicated DNA from the next origin; 15% of replicons are active at any given moment

Helicase

is the enzyme that separates DNA strands at the replication fork, and this enzyme requires ATP for its activity; multiple have been identified - in prokaryotes it is called DnaB

Single-Strand DNA Binding Proteins

; once the DNA is single-stranded, single-strand DNA binding proteins (SSBs) bind to it to keep it single stranded;

they bind cooperatively - meaning one binding helps the next one bind;

they may also be involved in recruiting DNA polymerase during DNA repair:

Topoisomerase

Introduces breaks into DNA allowing it to change ship

needed to change the shape of DNA

strand separation cannot occur without untwisting the DNA - DNA is not like a zipper with straight, parallel sides, but is a helix with strands

wound around each other

when the two strands of DNA separate, they must rotate around each other

•Closed circular DNA present special problems

-As DNA unwinds at one site more winding must occur at another site to reduce tension

Requirement for topoisomerases

Strands must unwind while they are separating

changes the topology of DNA

Negative supercoiling

(underwound,

less than 10.6 bp per turn)

Too few Turns

Positive supercoiling

(overwound, more than

10.6 bp per turn)

Too many turns

can prevent the polymerase from moving down the template if it is not corrected

Type 1 Topoisomerase

breaking one strand of double-stranded DNA

used to further relax negative supercoils, so these are not typically the ones needed during DNA replication

Type II Topoisomerase

DNA Gyrase in E. Coli

enzyme will bind one double-stranded part, and cut two strands, it will then pass another double-stranded section through it, relaxing the twisting

Cipro

works by blocking DNA gyrase activity in bacteria

DNA polymerase

adds triphosphate nucleotides to the replicating DNA by creating the phosphodiester bond

DNA Pol I

1). 5' to 3' polymerase activity - this is the main one we think about - it can add nucleotids to extend a 3' end - so it grows a new chain in the 5' to 3' direction

2). It has 3' to 5' exonuclease activity - this is also called "proofreading" activity - it can remove bases from the 3' end - this allows proofreading of mistakes made during replication - if a wrong base is added, it can be cut out and then replaced

3). It has 5' to 3' exonuclease activity - it can remove nucleotides from a free or nicked 5' end - this allows it to remove RNA primers - primer removal is the primary job of this particular enzyme; it is also involved in DNA repair

Klenow Fragment

: 5' to 3' polymerase activity and 3' to 5' proofreading activity

is used for primer extension DNA sequencing reactions... during sequencing, a primer is added and then you add Klenow fragment and nitrogenous bases that are labeled for detection (each nitrogenous base is labeled with a different color). Klenow will extend the primer and add labeled bases so that the sequence can be determined. You do not want the sequence to be degraded from the 5' end, which is why using just the Klenow fragment is a good choice for sequencing reactions. It can also proofread.

DNA Pol 1 Enzyme

Used for nick translation

5' ® 3' polymerase and 5' ® 3' exonuclease activity

both needed

The DNA pol I will use the 5' to 3' exonuclease activity to chop out bases from the 5' end

Then, DNA pol I will extend the 3' end at these empty regions by adding new nucleotides (some radioactively labeled) that are complementary to the opposite strand; DNA pol I cannot link the new fragment of DNA to the existing 5' end, so a nick remains. The nicks change location because of the activity of DNA pol I, which is why they called this procedure nick translation

DNase 1

Causes nicks in plasmid

DNA Pol 2

not needed for replication

Maybe a backup

DNa POl 3

3 subunits, 10 polypeptides

"replicase"

5' ® 3' polymerase activity

3' ® 5' exonuclease activity (proofreads

DNA polymerase d

replicates genomic DNA in eukaryotes

greatest processitivity

PCNA: clamps polymerase to template

Initiation

Requires a primer

Initiation In Prokaryotes

Occurs at the origin of replication

AT-rich

OriC: 9-mers = repeated regions

DnaA proteins are considered to be initiation factors for replication and will recognize the 9'mers and bind to the origin

DnaA

binding of DnaA recruits DnaB - DnaB serves as the helicase, and helps, along with HU protein, to separate the strands of DNA; HU protein has a structure similar to a histone, so it can bind to DNA and help to introduce negative supercoiling, making it easier for DNA to be separated