BIOL SCI 215 Study Guide - Midterm Guide: Exonuclease, Lac Repressor, Release Factor
28 Jun 2018
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DNA Structure and Replication
- Morgan’s model: genes are like beads on a string
- Gene sometimes called a “locus” (plural - loci)
- A location on a chromosome and its a particular stretch of DNA sequence
- Griffith’s experiments: Something can transform R cells into S cells
- Possible Inheritance
- DNA was the inheritance molecule / transforming agent
- Hershey- Chase experiment: virus bacteriophage DNA not protein is transferred into
infected cell
- Differentiate with radio-labeled atoms/isotopes
- Shows that radiolabeled DNA was transferred not proteins
- Chargaff’s rule - for each organism, A and T are in equal abundance as well as C and G
are in equal abundance
- Crystallography - DNA crystallized into lattices and a diffraction pattern is produced in
the X ray after they hit the sample, showing the 3D arrangement of atoms
- Suggests that DNA is a double helix through the diffraction pattern
- Overall structure of DNA (Deoxyribonucleic acid)
- Sugar phosphate backbone (outer part of DNA)
- Don’t need to know major and minor grooves
- Molecule adopts helix because of interaction between successive base
pairs
- DNA is a polymer composed of nucleotide monomers (nucleotide bases)
- Deoxy refers to lack of -OH hydroxyl group at 2’ position
- Hydrogen bonding in DNA composed of Purines (A,G 2 rings) and
Pyrimidines (C,T, U 1 ring), T and U are almost similar
- Deoxynucleotide Triphosphate (NEED TO KNOW), OH group at 3’
position
- 3 Phosphates connected to a 5-member carbon ring with a base
- Structure of four DNA nucleotides (Just need to know that it is a
nucleotide)
- Detailed structure of DNA
- Purine and Pyrimidines are able to hydrogen bond to each other
- Each Phosphate carries a negative charge and thus DNA is negatively
charged
- Each strand has a directionality
- Important features:
- Phosphate backbone is negatively charged
- Middle of double helix is hydrophobic
- Two strands held together by hydrogen bonding
- Anti-parallel notice 5’ - 3’ polarity on the strands
- Complementary strand of DNA
- Switch base pairs
- Melting temperature
- Heat will separate strands of DNA
- CG base pairs have 3 hydrogen bonds, AT has two hydrogen
bonds
- Longer strand = more hydrogen bonds
- Three models for DNA replication
- Semi- conservative
- Labeling experiment shows “semi-conservative” replication occurs
- Conservative
- New daughter strand has the same template as the parent strand
and just 1 out of 2
- Dispersive
- What needs to happen for DNA to replicate?
- Unwinding
- Through a molecule called heliocase
- Opens up DNA and a replication fork will be seen and then
synthesis will start
- Priming
- Synthesis
- E.Coli - broke it to release all proteins and then an assay was used to
measure synthesis
- Template DNA taken and they checked if that would catalyze DNA
synthesis
- Take complex mixture and purify it
- Properties of DNA polymerase
- Taking it, adding a template and see if DNA synthesis occurs - did not
work
- But when using a primer, they managed to get the reaction to work. It can
add to a growing chain but it cannot start from scratch
- Primer must bind, especially at 3’ end
- Important for base pairing to start DNA synthesis
- But a few mismatches tolerated at the 5’ end of primer
- You can go from 5’ to 3’ for the primer with regard to the new
strand
- ‘In vitro’ - experiment done in test tube
- DNA polymerization is ALWAYS 5’ -> 3’
- Line = single strand of DNA
- Directionality requires alternate strategy to replicate the two strands
1) Primase synthesizes short RNA oligonucleotides (primer) copied from
DNA
2) DNA polymerase elongates RNA primers with new DNA
3) DNA polymerase degrades RNA at 5’ end of neighboring fragment and
fills gap
4) DNA ligase connects adjacent fragments
Leading strand synthesis : Happens during unwinding
Lagging strand:
1) Helicase unwinds, melts DNA into 2 strands
2) Primase creates an RNA primer
3) DNA polymerase grows the chain
4) DNA polymerase degrades the RNA primer
5) For lagging strand synth, ligase joins together Okazaki fragments
Spots where replications occurs: Single origin/ Multiple origins
Prokaryotic: Circular Chromosomes, single origin - initiates a bubble of replication which allows
a continuous replication
Eukaryotic: Linear Chromosomes, Multiple origins (In between S phase, you have bubbles
showing multiple replications) Have problems with replicating the ends
Replication has a high fidelity: Relies on base parent
- DNA polymerase uses exonuclease activity to correct the mistakes made when pairing bases
and it is 3’ → 5’ (which is present in the protein)
4/30
Primers: Helicase, Primase, DNA pol, Ligase
- DNA is a polymer, its monomer is called dNTP
- Triphosphate is a high energy bond which helps to facilitate the reaction, taking 3’
Hydroxyl (from the base) and make a covalent bond
- Primer is made of RNA
- Lagging strand - with Okazaki fragment going backwards for 5’ to 3’, whereas for leading
strand, you can just continue
- End replication problem for linear chromosomes
- Solution: Generate arbitrary sequence which will add the base pairs to the ends
of the sequence using polymerase
- Telomere lengthening: RNA held within telomerase which is able to bind to the
base sequence. Enzyme does a reverse step making them complementary DNA.
Transcription in Reverse
- End is synthesized with primase
- Every eukaryote has a telomerase - important in cancer progression. WIthout it,
chromosomes will become smaller than coding genes will become lost
Document Summary
Morgan"s model: genes are like beads on a string. Gene sometimes called a locus (plural - loci) A location on a chromosome and its a particular stretch of dna sequence. Griffith"s experiments: something can transform r cells into s cells. Dna was the inheritance molecule / transforming agent. Hershey- chase experiment: virus bacteriophage dna not protein is transferred into infected cell. Shows that radiolabeled dna was transferred not proteins. Chargaff"s rule - for each organism, a and t are in equal abundance as well as c and g are in equal abundance. Crystallography - dna crystallized into lattices and a diffraction pattern is produced in the x ray after they hit the sample, showing the 3d arrangement of atoms. Suggests that dna is a double helix through the diffraction pattern. Sugar phosphate backbone (outer part of dna) Don"t need to know major and minor grooves. Molecule adopts helix because of interaction between successive base pairs.
