Pre-midterm material

Question 1

Describe backbone of nucleotide chain and it’s joining sites

Answer 1

Pentose sugar phosphate backbone
- 5’phosphate
- 3’hydroxyl - grows from this end

Question 2

Difference between RNA and DNA

Answer 2

DNA is missing 2’ hydroxyl on pentose sugar

Question 3

Describe the backbone of a protein and its ends

Answer 3

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

Question 4

What binds nucleotides? What binds amino acids?

Answer 4

• Link between nucleotides is phosphodiester bonds
• Link between amino acids is peptide bond

Question 5

Explain how nucleotides pair differently?

Answer 5

• G-C make 3 hydrogen bonds
• A-T make 2 hydrogen bonds

Question 6

In tertiary structure what is the type of bonds between side chains?

Answer 6

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

Question 7

Explain the Ubiquitin/proteasome degradation system

Answer 7

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!

Question 8

What is an amyloid?

Answer 8

Accumulation of misfolded proteins that plays a role in neurodegenerative disease, like Alzheimers or Parkinsons
These are often caused by mutations in ubiquitin

Question 9

Explain Michealis-Menten kinetics

Answer 9

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

Question 10

Explain centrifugation

Answer 10

• 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

Question 11

Explain SDS-page

Answer 11

Divides proteins based on mass
Denature proteins, and make then negatively charged - repelling each other

Migration rate is higher in smaller proteins

Question 12

Explain Isoelectric focusing

Answer 12

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

Question 13

Explain mass spectrometry

Answer 13

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

Question 14

Explain tandem MS

Answer 14

Fragmenting peptide ions by high energy collision with has, then doing MS spectroscopy on the fragments

Question 15

What are 3 types of chromatography? Explain each one

Answer 15

• 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.

Question 16

Explain the Western blot

Answer 16

Separation by weight using electrophoresis
- Electrophoresis followed by antibody detection

Question 17

Explain immunoprecipitation

Answer 17

Use of specific antibody to isolate a protein complex, which will be brought to bottom of column

Question 18

Name the molecules in DNA replication and what they do

Answer 18

• 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

Question 19

Explain DNA proofreading

Answer 19

• Epsilon and Delta polymerases proofread DNA

Question 20

Explain base excision repair, mismatch excision repair, and nucleotide excision repair

Answer 20

• 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

Question 21

Explain double strand break repair by end joining

Answer 21

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

Question 22

Explain homologous recombination

Answer 22

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

Question 23

Explain PCR

Answer 23

• 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

Question 24

Explain Sanger sequencing

Answer 24

Splitting DNA strand into 4 different mixers, each corresponding to a nucleotide

Put tubes through electrophoresis, this allows us to determine nucleotide sequence

Question 25

Explain NGS

Answer 25

New type of PCR based on single molecules
Minimize need for genome assembly, portable and allows reuse of sequences

Question 26

How does genome complexity change with complexity of organism?

Answer 26

There is more coding DNA in human genome than less complex organisms, however gene density is greater in lower eukaryotes

Question 27

Minisatellite vs microsatellite DNA

Answer 27

Microsatellite: 1- 4 bp in length, underlie neuromuscular disease
Minisatellite: 14 to 100 bp in length, 20-50 tandem repeat units, centromere and telomeres. Used for paternity tests adn criminal identification

Question 28

Retrotransposon vs DNA transposon

Answer 28

• Retrotransposon - cut and paste
• Transposon - copy and paste (increases copy number)

Question 29

What is a LINE? What encodes it?

Answer 29

LINE is a nonviral DNA retrotransposon (transposon that copy pastes itself)
- Has two open reading frames:
- ORF1: encodes RNA binding
- ORF2: encodes reverse transcriptase

Question 30

what is BLAST

Answer 30

Aids in finding nucleic acid and protein sequence similarity
Helps with aligns of proteins to find sequence similarity