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CHEM 430 > Lesson 16: Nucleic Acids Part 2 > Flashcards

Lesson 16: Nucleic Acids Part 2 Flashcards

1
Q

People involved in the early genetic evidence that DNA is genetic material

A

avery, mCcarty, Macleod

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2
Q

Avery, McCarty, and Macleod used the microorganism

A

Diplococcus
- 2 strains:

  • (R,rough) Aviruelnt
  • (S,smooth) Virulent

—-> they built on the work of F. Griffith

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3
Q

Griffith’s experiments

A
  • 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
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4
Q

Griffith’s conclusion

A

there is a transforming factor transferred from S to R cells

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5
Q

question that arose from griffith’s conclusion

A

What was the transforming facor??? DNA?RNA?Protein?

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6
Q

Avery et. al experiment 1994: DNA, RNA, protein?

A
  • 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

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7
Q

Avery et. al experiment 1994 results:

A

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

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8
Q

more direct evidence DNA is the genetic material: Hershey and Chase, 1952

A

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

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9
Q

Phage DNA life cycle

A
  • 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
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10
Q

Hershey and Chase experiment

A
  • 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
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11
Q

secondary structure of nucleic acids

A
  • 3D arrangements of nucleotide residues with respect to one anothe, and short-term folding interactions such as the double helix
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12
Q

tertiary structure of nucleic acids

A
  • longer range 3-D interactions such as supercoiling, superhelical forms and higher orders of “packing”
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13
Q

double helix-experimental evidence - base composition studies: edwin chargaff 1952

A
  • 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
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14
Q

chargaff’s rules

A

a = t
g = c
a + g /t + c = 1
a + t / G + C does not = 1

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15
Q

X-ray crystallograpgy: rosalind Franklin

A
  • demonstrated the “periodicity” of the double helix
  • proposed bases inside and phosphates outside
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16
Q

characteristics of double helix

A
  • 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
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17
Q

3 main DNA conformations

A
  • 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
18
Q

B-DNA and A-DNA base pairing requires strands to be antiparallel/parallel for canonical watson-crick base paring

A

antiparallel

19
Q

reverse base pairing can be demonstrated in vitro;

A

H -bonding pattern in reverse W-C base pairing is different

  • less stable than normal base pairing
20
Q

what kind of helicies are B-DNA and A-DNA

A

right handed helicies

21
Q

where is B-DNA found

A

under physiological conditions (pH; temp)

22
Q

how can A-DNA be formed

A

by dehydrating B-DNA

23
Q

canonical watson-crick base pairing

A
  • 3 base pairs between G and C
  • 2 base pairs between A and T
24
Q

in the B-form DNA, the base pairs are stacked, but in the A-form…

A

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

25
Q

major groove of DNA helix

A

result of 2 polynucleotide chains wrapping around themselves in a right handed fashion yielding B-DNA
- a lot of base pairs visible for transcription

26
Q

minor groove of DNA helix

A

fewer base pairs for transcription factor to come in and interact/read information

27
Q

AU and GC H-bonding as well as non Watson-crick base pairing leads to

A

base stacking

28
Q

are linear molecules favored under cellular conditoins

A

NO – due to the conformational entropy and the crowded interior of cells

29
Q

forces that stabilize nucleic acid structures

A

1 - hydrogen bonding and base stacking (base stacking is the primary stabilizer)

2 - AT base pairs and GC base pairs

30
Q

base stacking to stabilize

A
  • 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
31
Q

base stacking stability is sequence specific

A

most stable; lower energy
– G/C base combinations

least stable: higher energy
– less energy required to disrupt A &T bonds

32
Q

nucleic acids can be reversibly denatured

A
  • thermal denaturation, or melting, can be used to determine stability
33
Q

hyperchromic shift

A

the increase in A360 as dsDNA –> ssDNA

34
Q

Tm =

A
  • temperature where 50% DS 50% SS
35
Q

why does single stranded nucleic acid absorb more UV light than double stranded

A
  • 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
36
Q

how does ionic strength affect Tm

A
  • 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
37
Q

how does G-C content affect Tm

A
  • 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
38
Q

higher G-C content,

A

higher Tm
- more stacking dimer, more E to melt out stacked dimers

39
Q

lower G-C content,

A

meaning higher A-T content
- lower stacking energy, meaning lower Tm

40
Q

the Tm of ncleic acids

A

CHEM 430 (23 decks)

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