Describe backbone of nucleotide chain and it’s joining sites
Pentose sugar phosphate backbone
- 5’phosphate
- 3’hydroxyl - grows from this end
Difference between RNA and DNA
DNA is missing 2’ hydroxyl on pentose sugar
Describe the backbone of a protein and its ends
Protein backbone is a Carbon with a carboxyl on one side and Amino group on the other
Grows by addition of monomers to Carboxyl end
What binds nucleotides? What binds amino acids?
- Link between nucleotides is phosphodiester bonds
- Link between amino acids is peptide bond
Explain how nucleotides pair differently?
- G-C make 3 hydrogen bonds
- A-T make 2 hydrogen bonds
In tertiary structure what is the type of bonds between side chains?
Mainly non-covalent (H-bonds, ionic bonds…)
There is ONE important covalent bond - cysteine side chain contains sulfhydryl that creates covalent bond with other cysteines
Explain the Ubiquitin/proteasome degradation system
Ubiquitin is a protein that marks uneeded proteins as “trash” for proteasome to recognize
Proteasome removes ubiquitins and unfolds target protein with ATP, feeds it into 20S. Unfolds misfolded proteins!
What is an amyloid?
Accumulation of misfolded proteins that plays a role in neurodegenerative disease, like Alzheimers or Parkinsons
These are often caused by mutations in ubiquitin
Explain Michealis-Menten kinetics
Graphs
Vmax - rate of catalysis given S - saturating amounts of substrate
Km - substrate concentration that supports a rate of catalysis equal to 1/2 the Vmax
4 times as much enzyme in reaction gives Vmax 4fold higher
Explain centrifugation
- Rapid spinning of a centrifuge tube - generating centrifugal force
Forms pellet at bottom of tube and rest of liquid is supernatant
If particles are less dense than medium, they float at top of tube
Can create a density gradient and place pellet in it, to determine density of pellet
Explain SDS-page
Divides proteins based on mass
Denature proteins, and make then negatively charged - repelling each other
Migration rate is higher in smaller proteins
Explain Isoelectric focusing
Measure of sum of all charges rather than weight
- isoelectric point determeined by amino acids composition of protein
- negatively charged go to low pH and positive charge go to high pH - protein will resolve at it’s isoelectric point
Explain mass spectrometry
Determination of charge-to-mass ratio
- produce dispersed ions in a gas phase, measure their acceleration in an electric or magnetic field, allowing us to determing their charge to mass ratio
Explain tandem MS
Fragmenting peptide ions by high energy collision with has, then doing MS spectroscopy on the fragments
What are 3 types of chromatography? Explain each one
- Gel filtration: separation based on size. Protein is placed in solid gel beads. Large proteins exit the column first, while small proteins filter into beads
- Ion-exchange chromatography: separation based on electric charge. Place cation or anion in column as immobile phase, proteins with net electric charge will flow through first
- Antibody-affinity chromatography: Bases on antibodies recognizing epitope (molecular target). Antibodies recognize epitope. Column us loaded with epitopes, and run antibodies through column. Desired antibodies will stick to epitopes and remain in column.
Explain the Western blot
Separation by weight using electrophoresis
- Electrophoresis followed by antibody detection
Explain immunoprecipitation
Use of specific antibody to isolate a protein complex, which will be brought to bottom of column
Name the molecules in DNA replication and what they do
- Rep protein A: binds single-stranded DNA, keeps it in optimal conformation
- DNA Pol epsilon: carries out leading strand DNA synthesis
- PCNA: prevents Pol epsilon from disassociating from the template
- Primase/Pol alpha complex: lays primer and extends primer with DNA
- Pol delta/RFC / PCNA complex (4 per replication bubble - 1 per fork): replaces pol alpha and completes okazaki fragments synthesis, RFC opens PCNA rings and loads it as a primer on DNA, pol delta replace RNA with DNA
- RIbonuclease H and FEN-1: displace RNA at 5’ end of okazaki fragment
Explain DNA proofreading
- Epsilon and Delta polymerases proofread DNA
Explain base excision repair, mismatch excision repair, and nucleotide excision repair
- Base excision repair: damaged base (cytosine is often deaminated to form uracil). Use DNA pol B to break off backbone and fill gap
- Mismatch excision repair: incorrect base in strand. Gap repair by Pol delta and DNA ligase after cut by endonucleases
- Nucleotide excision repair: a modification in normal shape of DNA. Repaired with formation of thymine-thymine dimer to help recognize site, XP factors cut the bump, and pol d repairs the gap
Explain double strand break repair by end joining
Problem: Double strand break due to ionizing radiation (often anticancer drugs)
Highly error prone that often introduces new mutations
1) Kinase heterodimer binds to ends of break
2) Ends digested by nucleases
3) Ends ligated together
Explain homologous recombination
1) exonucleases digest the ends of the break
2) NEED TO HAVE HOMOLOGOUS CHROMOSOME AVAILABLE FOR THE REPAIR
3) Rec A and Rad51 begin invasion of homologous chromosome
4) DNA pol extrans 3’ end of invading strand
5) 5’ ends ligate to form Holliday junction
6) Structure are resolved - 4 knicks are sealed total
Explain PCR
- Need Taq polymerase, DNA template, primers, and dNTPs
- DNA is boiled, separating its strands and complementary strand is made using primers and dNTPS
This is repeated, creating a large amount of DNA fragments
Explain Sanger sequencing
Splitting DNA strand into 4 different mixers, each corresponding to a nucleotide
Put tubes through electrophoresis, this allows us to determine nucleotide sequence
