1L1 DNA Structure & Organization

Which experiment identified DNA as the genetic material?

Avery, MacLeod, and McCarty (1944).

***AvEry Extracted the sEcret

Which experiment confirmed DNA is the genetic material using bacteriophages?

Hershey and Chase (1952).

***Hershey chases virus

What experiment revealed DNA’s double helix structure?

X-ray diffraction by Rosalind Franklin and Maurice Wilkins,

interpreted by Watson and Crick (1953).

central dogma

DNA + transcription > RNA + translation > protein

DNA > 2DNA via replication

prokaryotes v eukaryotes

DNA in cytoplasm v. DNA in nucleus

DNA

deoxyribonucleic acid (missing 2’ OH)

avery’s experiment

What type of molecule contains the genetic information of a cell?

Streptococcus pneumoniae

• Smooth cells produce a polysaccharide capsule, rough cells don’t

• bacterial transformation

bacterial transformation

bacteria takes up external DNA to change morphology and physiology

cell-free extract

S cell heated to kill, fragments released as extract

extract is mixed with live R cells, R > S via transformation

proteinase v rnase v dnase

degrades protein R > S

degrades RNA R > S

degrades DNA R X S

nucleotides

nucleoside + (1-3)P = nucleotide

purine structures

2 rings

pyrimidine structures

1 ring

polynucleotides

DNA and RNA, 3’OH attacks 5’P and connects nucleotides (phosphodiester bond)

3’ end = free OH

chargaff’s rules

1. % A=T and G=C

2. Base composition varies between species. (human = 59% AT, yeast = is 64% AT)

3. Base composition is the same in different cells within an individual organism.

4. Base composition does NOT change with age, nutrition, and environment

DNA v RNA structure

2’ OH is missing in DNA

Photo 51

previously only photos were of dehydrated DNA

xray diffraction to show location of atoms,

shows helix 34A long, 10 base pairs 3.4A apart

dNMP

2'-deoxyribonucleoside 5'-monophosphates

repeating unit of DNA

2D DNA structure

2 unbranched polynucleotide chains, antiparallel, held by H bonds

watson-crick-franklin base pairing = AT GC

sugar and phosphate backbone

DNA strand separation & annealing

denaturing/melting DNA (Tm = 50% denatured)

when cooled, DNA will anneal back together

3D DNA structure

BDNA = 99% of DNA

• right handed helix, 10.5 base pairs per turn

• base pairs flat and perpendicular to backbone

• Hphobic bases inside

• base pairs exposed in major and minor grooves

ADNA v ZDNA

ADNA = dehydrAted, structural model of dsRNA and RNA-DNA hybrids

ZDNA = <1% DNA, high GC content, left handed

cruciform, triplex, quadruplex

RNA structure

linear, single stranded

• ribose sugar P backbone

• U instead of T

• GU wobble base pairing = RNA pairs with RNA

RNA complex structures

secondary and tertiary from internal base pairing

gene, genome, chromosome

gene contains DNA and regulatory elements

genome is all of the genes

46 chromosomes = helps position and organize genes in the genome

polynucleotide synthesis

always 5 > 3, NTPs as substrates

DNA polymerase = adds dNMP to 3’ end of DNA chain, requires primer

RNA polymerase = adds NMP to 3’ end RNA chain, can initiate de novo synthesis

reverse transcriptase = adds dNMP to 3’ end of DNA chain, requires primer

DNA polymerase

adds dNMP to 3’ end of DNA chain, requires DNA primer

RNA polymerase

adds NMP to 3’ end RNA chain, can initiate de novo synthesis

needs DNA template

reverse transcriptase

adds dNMP to 3’ end of DNA chain, requires primer and RNA template

nucleases digest polynucleotides

exonuclease = breaks Pdiester bond at one end

endonuclease = breaks Pdiester bond within chain

excinuclease = breaks 2 Pdiester bonds in single chain

exonuclease

breaks Pdiester bond at one end

endonuclease

breaks Pdiester bond within chain

• sequence independent or specific

• single strand (nick) or double-strand break

excinuclease

endonuclease; breaks 2 Pdiester bonds in single chain

restriction endonucleases / enzymes

endonuclease that breaks PDE bonds at specific restriction sites

palindromic sequences = same on both strands, forms sticky ends when cut

DNA ligase

links two existing DNA chains via PDE linkage

1. grabs AMP from ATP or NAD+

2. adds onto itself to activate

3. scans DNA for substrate (nick)

4. adds AMP to 5’P of DNA as a good leaving group (GLG)

5. 3’OH attacks 5’P, forms PDE bond, remove AMP

6. bond is sealed

in vivo, seals nicks

recombinant DNA

new combos of DNA

restriction enzymes isolate new DNA, cut old DNA to match sticky ends, and DNA ligase fixes PDE bonds to form singular DNA

AT v CG bonds

AT has 2, CG has 3

DNA detailed structure

3’OH forms PDE bond with 5’ phosphate in the ribose backbone,

1’ bases H bond together