Chem Bio test 1 Flashcards
Common Chem Bio tools?
Chromophores, Assays, Microbiological Screens and Viruses
Some Small Molecules that AFfect Pathways (DNA->Proteins)
Aphidicolin, Alpha Amanitin, cyclohexamide, alendronate, platensimycins, soyasaponins
What determines bonding?
Coulomb’s law, sterics, filled vs unfilled orbital overlap
Why is nucleophilic attack on phosphate esters slow relative to carboxylic esters?
High cost to rehybridize, presence of electronegative alkoxy substituents and repellant from the negative charge.
Effect on stability of the 2’OH of RNA?
DNA less likely to undergo base promoted phosphodiester bond cleavage
but
RNA less likely to undergo SN1 acid promoted solvolysis of the nucleobase
Pros and Cons of Small molecules
Pros: Membrane permeability,
Cons: low selectivity, difficult to synthesize
PCR reagents
dNTP’s, template, primers, MgCL2, polymerase
What’s the problem with bromo-uracil?
0.1-1% of bromouracil is in the ENOL form and can base pair with Guanine (mismatch) instead of adenine
Interactions that would help something bind to DNA
+ charge, arene activity (intercalators), curved shape to fit along DNA, epoxides (ring strain)
Examples of how transcription is controlled?
Availability of DNA, Protein modification (histones, repressors, transcription factor binding), regulatory nucleic acids that make secondary structures or block binding
How is translation controlled?
Small molecule inhibition of ribosome, mRNA degradation,
The reversibility of non-bonding interactions
Kinetic Instability
Molecular oxygen is most stable in singlet form because the lone pair oxygens are paired
false
Pi stacking is strongest when the rings are right on top of each other
False
What are synthetic base pairs admitted on?
Sterics (not h-bonding - don’t need h-bonding)
What wavelength do we monitor to see unfolded vs folded DNA
260 nm (should see an increase in absorbance when unfolded
Where are the fluorophores attached for ddntps
The 3 or 8 nitrogens
Characteristics of error prone PCR?
High DMSO content, low fidelity polymerase or Mn2+
Difference between ddNTP’s and dNTP’s
dd is deoxy on 2 AND 3 so - can’t continue PCR
A pair of negatively charged functional groups and a pair of neutral atoms are initially 2 Angstroms apart and then are moved to 3 angstrom apart from each other (each unit in a pair). Describe how the energy of their interaction changes?
The negatively charged are repulsed and the change in repulsion is proportional (so tit is now 2/3’s of the original - decreased by 1/3). For the neutrals - they have a slight attraction initially from van der waal forces and this DECREASES by MORE THAN 90% - a very drastic change.
Reverse Transcript can use DNA or RNA as primer?
Both
What is on our plasmid
ORI, Promoter, operator, terminator, selectable genetic marker, info for transcribing the repressor, Multiple cloning site, any tag for affinity purification, encoding translation (RBS)
Cloning steps?
Generate recombinant vector (PCR), use restriction enzymes to introduce
Introduce to organism (take up)
Kill off those that didn’t take it up
Express protein
Purify
Major RNA structural motifs
Coordinating divalent cations (diffuse and site dependant), HOOGSTEIN triple base pairing, deep major groove, shallow minor groove, short herlices, base pairing in lines with the helix (as opposed to through the center), long distance BP
Difference between acid catalyzed and base catalyzed phosphodiester cleavage in RNA
In Acid - The phosphate is protonated and more stabilized - allows for the rearrangement to make it a 2’ OH, - in Base - not the case - just end up cleaving the 5’ bond on the next nucleobase
Common targets in Thymine synthesis
Step 1 - Removal of 2’OH and Step 3 - Removal of Methyl
Most nucleophilic Nitrogens across DNA base pairs
G (N7), A(N3), A(N1), C(N3)
Cation pi interactions are destabilziing
False
Becaose of the SP3 hybridization of water - 109.5 degrees is the strongest H bonds
False
Amides are more stable than esters because of resonance
True!
Key Reaction in translation
Aminolysis of ester bond to amide (catalyzed by weak bases)
Which nucleotide performs the key step of translation
N3 of Adenine
Interaction types to look out for?
Coulombic, Van Der Waals, Dipole, Hydrophobic, Pi stacking (arene activity). Positive charge ( for negative charge for DNA)
Where does Hoogstein base pairing occur in DNA
Major groove (DOES OCCUR!)
Order of DNA transcription events
Transcription begins RNA POLII binds - once 5’ mRNA is out - it gets capped - then splicing occurs at splcie points then finally when done - poly A tail at 3’ end
How does tRNA synthase recognize the right tRNA
Global conformation and induced fit
What causes the color change in 4-nitrophenol?
Loss of aromaticity through resonance
Describe how DNA replication begins with RNA primers
DNA primase lays down RNA primers because DNA polymerase can only extend. It extends and eventually RNA-ase comes back and chews it up and it’s replaced by DNA polymerase and DNA ligase to connect the two
Steps in DNA polymerase error correction
If the wrong base pair is present and the reaction proceeds - due to poor fit - it will occur at a much slower rate and causes a different pathway where an exonuclease to cut it out
What are common checkpoint failure that can lead to apoptosis
Not enough DNA or too much DNA repair
common RNA 2ndary structures
Hairpins and stem loops
What makes up energy of bonding
Charge, reuplusion attraction (Charge is R^-1), repulsion is R^-6) and attraction is R ^-12)
Longevity of DNA , RNA ,peptides lipids
That order
pka of phosphodiester
1-2
Why does RNA undergo phosphodiester rearrangement under acidic conditions
Under acidic conditions - the phosphate can be protonated - make it more neutral -
What are the 3 amino acids important in RNA degradation
His 119 (protonated - acts as acid - stabilizes)), His 12 (deprotonated acts as base - removes H from OH)) and Lys 41
