Lesson 16: Nucleic Acids Part 2 Flashcards
People involved in the early genetic evidence that DNA is genetic material
avery, mCcarty, Macleod
Avery, McCarty, and Macleod used the microorganism
Diplococcus
- 2 strains:
- (R,rough) Aviruelnt
- (S,smooth) Virulent
—-> they built on the work of F. Griffith
Griffith’s experiments
- had a smooth (virulent) colony (IIIS) and a rough (avirulent) colony (IIR), then injected both into the mouse
- the mouse died in the virulent colony but lived i the avirulent colony
- then he injected heat-killed IIIS into the mouse, and the mouse lives
- (CRITICAL EXPERIMENT) then he injected living IIR (rough,avirulent) and heat killed IIIS (smooth/virulent) into the mouse and the mouse died
^^^^ tissue was analyzed and he found living IIIS
Griffith’s conclusion
there is a transforming factor transferred from S to R cells
question that arose from griffith’s conclusion
What was the transforming facor??? DNA?RNA?Protein?
Avery et. al experiment 1994: DNA, RNA, protein?
- took heat killed IIIS filtrate (contains DNA, RNA, and proteins) then treated it w/ 3 differnt things
1 – control: adding living R cells with the head killed S cells containing DNA/RNA/proteins
2 – treat w/ protease – living R cells with protease-treated IIIS filtrate
3 – treat w/ riibonuclease – living R cells with RNase-treated IIIS filtrate
4 – treat w/ deoxyribonuclease – living R cells + DNase-treated IIIS filtrate
Avery et. al experiment 1994 results:
1 —control –> transformation occurs – living avirulent cells and now living virulent cells – MEANING IIIS contains active factor
2 – > transformation occurs – found living R cells and living S cells — MEANING active factor is not a protein
3 –> transformation occurs – found living R cells and living S cells — MEANING active factor is not RNA
4 –> no transformation occurs – only living R cells – MEANING active factor is DNA
more direct evidence DNA is the genetic material: Hershey and Chase, 1952
1 - utilized radioactive isotopes of phosphorus (P32) and sulfur (S35)
2 - P is found in DNA and RNA, S is found in amino acids: Met and Cys
Question asked: Does DNA or protein enter the host cell? —> Whatever enters the host cel must direct the virus lifecycle
Phage DNA life cycle
- phage attaches to bacterium
- phage genetic material is injected into bacterium
- phage reproductive cycle begins
- components accumulate; assembly of. mature phases occurs
- cell lysis occurs and new phages are released
Hershey and Chase experiment
- took Phage T2 (unlabeled) and placed it in a flask of E coli in radiactive medium (either 32P or 35S)
- phage self labeled witheither 32P (DNA) or 35S (protein)
- isolated labeled phage and infect a bacterium
- allow phages to infect then separate “ghosts” from infected host cells
- 32P in pellet; therefore DNA entered the host cell
- 35S in sup; therefore protein does not enter the host cell
secondary structure of nucleic acids
- 3D arrangements of nucleotide residues with respect to one anothe, and short-term folding interactions such as the double helix
tertiary structure of nucleic acids
- longer range 3-D interactions such as supercoiling, superhelical forms and higher orders of “packing”
double helix-experimental evidence - base composition studies: edwin chargaff 1952
- measured the mole fraction of each base per mole of phosphate (remember the components of a nucleotide)
- base composition of NDA generally varies between species
- DNA from different tissues of the same species have the same base composition
- base composition of DNA in a species does not change with age, environment, etc.
- in all cellular DNA;s reguardless of species, A=T; G=C, A+G = T+C
- provided initial insight into how bases interact with each other –> base pairing stoichiometry
chargaff’s rules
a = t
g = c
a + g /t + c = 1
a + t / G + C does not = 1
X-ray crystallograpgy: rosalind Franklin
- demonstrated the “periodicity” of the double helix
- proposed bases inside and phosphates outside
characteristics of double helix
- hydrogen bonding between purines and pyramidines
the distance between bases is 3.4 angstoms
- the diameter of the helix is 20 angstroms
- there are 10 base pars per turn of the helix in B-DNA (34angstroms/turn)
- there are 3 main DNA conformations
3 main DNA conformations
- B-DNA: right handed double helix
- 2 strands are anti-parallel
- major and minor groove
- A form DNA = largest diameter, them B-form, then Z-form
B-DNA and A-DNA base pairing requires strands to be antiparallel/parallel for canonical watson-crick base paring
antiparallel
reverse base pairing can be demonstrated in vitro;
H -bonding pattern in reverse W-C base pairing is different
- less stable than normal base pairing
what kind of helicies are B-DNA and A-DNA
right handed helicies
where is B-DNA found
under physiological conditions (pH; temp)
how can A-DNA be formed
by dehydrating B-DNA
canonical watson-crick base pairing
- 3 base pairs between G and C
- 2 base pairs between A and T
in the B-form DNA, the base pairs are stacked, but in the A-form…
there is a 20 degree roll/tilt (as opposed of being perpependicular
- a change in slide causes tha base pairs to shift away from the axis of the helix
- a change in roll causes the plant of each base pair to tilt by about 20 degrees
major groove of DNA helix
result of 2 polynucleotide chains wrapping around themselves in a right handed fashion yielding B-DNA
- a lot of base pairs visible for transcription
minor groove of DNA helix
fewer base pairs for transcription factor to come in and interact/read information
AU and GC H-bonding as well as non Watson-crick base pairing leads to
base stacking
are linear molecules favored under cellular conditoins
NO – due to the conformational entropy and the crowded interior of cells
forces that stabilize nucleic acid structures
1 - hydrogen bonding and base stacking (base stacking is the primary stabilizer)
2 - AT base pairs and GC base pairs
base stacking to stabilize
- hydrophobic bases stack on top of each other
- water molecules exlcuded; increases entropy of solution
- when bases are at van der waals distance; it maximizes interactions between the rings; enthalpic stability through maximum of weak interactions formed
base stacking stability is sequence specific
most stable; lower energy
– G/C base combinations
least stable: higher energy
– less energy required to disrupt A &T bonds
nucleic acids can be reversibly denatured
- thermal denaturation, or melting, can be used to determine stability
hyperchromic shift
the increase in A360 as dsDNA –> ssDNA
Tm =
- temperature where 50% DS 50% SS
why does single stranded nucleic acid absorb more UV light than double stranded
- SS DNA: N-bases now solvent exposed therefore greater UV absorption
- DS DNA: N-bases interacting with each other; less solvent exposed therefore interacting lower UV absorption
how does ionic strength affect Tm
- low ionic strength leads to increased electrostatic repulsion and decreased Tm
- high ionic strength leads to ion pairing with negative backbone, which increases Tm
- increase of Na ions interacting w/ backbone of DNA, masking the negative charge making it easier to destabilize, BUT you still need to melt the ionic interactions, thus Tm increases
how does G-C content affect Tm
- Tm increases as duplex lenth increases at constant G-C and [NaCl]
- increased stacking interactions, pushing Tm value to the right
- if you increase the humber of base pais than you are increasing the amount of energy as heat that is required to melt down 50% of double stranded DNA molecules
higher G-C content,
higher Tm
- more stacking dimer, more E to melt out stacked dimers
lower G-C content,
meaning higher A-T content
- lower stacking energy, meaning lower Tm
the Tm of ncleic acids