23 & Me
Who discovered that DNA is a helix?
Rosalind Franklin
Why are origin of replication sites rich in “AT” base pairs?
Easier to break apart than GC
Why isn’t an RNA primer used in PCR?
The RNA primer would need to be replaced and removed at the end, plus RNA is less stable than DNA.
Presence of chromatin in eukaryotic DNA increases melting temp.
What is the core unit of chromatin?
Nucleosome
What is Chargaff’s rule?
There’s an equal number of purines and pyridimines
(A+G = T+C)
What are the necessary ingredients for PCR?
Forward and reverse primers
Taq DNA polymerase
All 4 dNTPs
Template DNA
Why do we use ddNTPs in Sanger sequencing?
ddNTPs do not have a C3’ hydroxyl group, so it acts as a chain terminator
Where does transcription start?
Semi-conserved regions called promoters
What are the requirements/ingredients for RNA synthesis?
Holoenzyme, Mg2+, DNA template, ATP/GTP/CTP/UTP
What is the primary stabilizing effect in DNA?
Pi-stacking
What is a Rho factor?
Rho is a transcription termination factor in prokaryotes. It binds to specific regions of the transcribed single-stranded RNA and inhibits transcription.
What’s the purpose of Mg2+ ions in DNA synthesis?
Used to coordinate the 3’-OH of primer and alpha-phosphate of incoming dNTP
What is the equilibrium of DNA synthesis driven by?
It’s driven by the relative binding energy of bases
Why is telomerase a reverse transcriptase?
It is synthesizing the DNA in the 5’ to 3’ direction using RNA as a template (instead of DNA)
Why was Pauling’s model of DNA incorrect?
It had the bases on the outside and phosphates in the middle, instead of vice versa. Ignored hydrophobic effect. Also ignored Chargaff’s Rule.
Describe the difference between conservative, semi-conservative, and dispersive models of replication. Which does DNA use?
DNA use semi-conservative - each daughter DNA gets one new strand and one old strand.
Conservative - one daughter DNA is all new, the other is all old
Dispersive - each daughter DNA is a mixture of old and new.
Which DNA Polymerase has 5’ → 3’ exonuclease activity, as well as 3’ → 5’ exonuclease activity.
How does primase fit in with the RNA world hypothesis?
Primase is an RNA primer that initiates DNA synthesis. It fits with the hypothesis because it shows RNA synthesis is essential for DNA synthesis, so RNA would have come first.
Name two reactions catalyzed by ribozymes.
-phosphodiester cleavage
-phosphate transfer
-peptide bond synthesis
What type of reaction is the 3’ → 5’ exonuclease reaction?
Hydrolysis reaction. It’s a two-metal-ion mechanism, used for recognizing mismatches.
How did Meselson & Stahl’s experiment support semi-conservative mode of replication?
They first grew E. coli in 15N media and then switched over to 14N. After a number of generations, they stopped the growths and extracted & centrifuged the DNA with CsCl They found that after one generation, the DNA band was shifted to exactly between 14N and 15N labeling and then after two generations there was a new 14N band in addition to the band between the 14N and 15N densities. After more generations, the 14N band grew in intensity but the band between the 14N and 15N band stayed the same.
Give 2 ways that Mg2+ is used in DNA Pol 1 active site.
-Assists in deprotonation of 3’ OH base
-Stabilizes pyrophosphate, making it a better leaving group
-critical component in organization of reaction partners
In the final step of DNA synthesis, DNA ligase repairs the nicks. Why is the 5’ phosphate adenylated at the nick?
Need a good leaving group attached to the electrophile to make it more electrophilic
TATA-Binding Protein (TBP) is involved in DNA melting to start transcription. How does it do this?
TBP bends DNA by about 80 degrees by inserting Phe residues into minor groove. This forces the minor groove open and helps it melt.