Lecture 28: Nucleic Acids Flashcards
DNA: where are bases? backbone?
bases: center of helix
backbone: outside
stability of double helix structure due to what?
BASE STACKING
NOT base bonding
base pairing
done by hydrogen bonding between pairs
this adds little stability
form of RNA double helicies
A form
stems and loops (heleices and signel stranded RNA that arent base paired)
what pairs with what?
G-C
A-T
If one DNA strand has sequence 5’ATCGGCA3’, what is sequence of COMPLIMENTARY strand?
5’TGCCGAT3’
start from BACK because ANTIPARALLEL
what is base PAIRING important for?
SPECIFICITY
not stability
how is DNA written?
ALWAYS 5’ to 3’
how do bases associate in hydrophobic environments?
by hydrophobic interactions
Base Stacking
chemistry and attractions for each other van der Waals interactions hydrophobic interactions (shielding from water=attraction) stability of helix
Stacks
aromatic rings, parallel ring systems above and bellow
interact by van der Waals interactions
how many hydrogen bonds between AT?
2
how many hydrogen bonds between GC?
3
Why is it wrong that GC bonds are stronger than AT bonds?
really, its Gs stacked on Cs are much stronger than As stacked on Ts
stacking
need two base pairs in a row:
C-G
G-C
=-61 kJ/mol (inverse is only -40)
A-T
T-A
-16 kJ/mol inverse is -27.5)
WHY is C over G stronger than A over T?
idk!
why is it base stacking not pairing?
It can’t be hydrogen bonding because energy is different for G on C than it is for C on G (also for As and Ts)
same number of hydrogen bonds, but different energies
Thymine vs. Uracil
both pyrimidines that pair with adenine (purine)
DNA has A-T
RNA hybrids with DNA have (RNA)U-A(DNA) or (DNA)A-T(DNA)
Why does RNA contain U but DNA contains T?
spontaneous deamination of cytosine makes uracil
this happens constantly
Why can’t we have U in DNA?
if we had uracil in DNA, it would be really easy for G-C pairs in DNA double helix to mutate to TA after TWO rounds of replication
How do we avoid the problems with uracil in DNA?
enzymes evolved to quickly remove uracil from DNA b/c it doesn’t belong
Us NOT ALLOWED IN DNA!!!!!!!
why does it take 2 rounds of rep for a mutation from GC to TA?
at first: the C won’t pair with the U
first round, it will just put an A with the U instead of the C
second round: then we get put a T with the A
the three forms of DNA helix: A, B, Z
all same sequence, just differing interactions
B is most common
best for stability and stacking and shielding
how do A and B forms differ?
A is more squished down
stacking: why are stacks between same base pairs (C-G vs G-C for example)?
stacking is more stable due to HOW its stacked, how top of one interacts with bottom of other
Hybridizing through base pairing
determined by base PAIRING not stacking
naturation and renaturation
heated up, unravelling, getting back together
Unravelling…
relative to the STACK
what’s cool about nucleic acid helix separation?
doesn’t break covalent bonds
only non-covalent weak interactions are disrupted (van der Waals and hydrogen bonds)
What is more/less stable?
GC stacks more stable than AT
review slide 11
review slide 11
Using spectroscopy to determine number of strands
measure absorbance of nucleotide bases
use wavelength of 260nm, you can distinguish bases
absorbance for double stranded/signle stranded
low absorbance for double stranded (bases inside) (but increases as it unravels)
high absorbance for single stranded (bases stick out)
what does “cooperativity of transition” mean?
LOOK THIS UP!
It is an in-between state of pulling apart/denaturation. The midpoint of the S curve is the point where it goes from being wound to completely denaturing
what is the significance of the sigmoidal curve on the graph?
DNA is well ordered at low temp, it opens up as temp incs
when we get to critical temp, the DNA just pulls apart b/c stacking can no longer hold it together
What is the composition of the DNA pool at 85 degrees celcius. Are most DNA strands 50% single stranded, or are 50% of the strands completely single stranded and 5o% of the strands completely double stranded?
half of small DNA molecules are double stranded, half are double stranded
b/c they are small molecs (long molecs would cause “bubbles”
What affects the “melting temperature” (Tm) (the transition temp)
base composition
salt (NaCl),
length of complementary sequence
What happens if we change ratios of Gs and Cs?
Tm increases with inc precentage of G-C content
b/c G-C base stacking interactions are more stable in double helix
Is the localized Tm affected by the specific DNA sequence? Explain answer in terms of base stacking nrgs
(ex CAGTCTGA vs. ATTAGCCG)
how is this biologically useful
Yes
As and Ts at the bottom, pulls apart faster in this part in second strand
The more GC, the higher the temperature needs to be to pull it apart. The second would pull apart faster b/c there is lower stacking energy for AT/TA.
biologically useful if you want a bubble and want a protein to sneak in
How does length affect Tm
lower temp needed for shorter strand
but really only matters if under 70
Length of DNA only affects Tm for DNA fragments less than about 70 bp. Why?
melts quicker if shorter (up to a certain length)
Considering that DNA forms a helix in pure water, do you want to store DNA in water?
NO! you want so salt in there
Salt content of cell… how does Tm get affacted?
Sodium ions neutralize negative charges and STABILIZE the helix
so more salt=higher temp needed to break it down
why is it bad to store DNA in water?
there are no ions!!!
DNA way less stable
why? at low salt, we barely shiled the phosphates
DNA palindromes
evolutionarily selected for functions 2-fold symmetry self complimentary and form hairpins repeat self uses for sequence, not structure
RNA single and double stranded regions
RNA is frequently base paired to make double strands
Transfer RNA
adaptor in protein synth
folds to complex tertiary strucutre, about half nucleotides are paired
some modified tRNA bases
Protein binding
bind to DNA using sequence specific fasion
critical in gene regulation
required for DNA replication, repair, and recombination
protein affinity is really high
involve non-covalent weak interactions
most common H bond donors:
Asn Gln Glu Lys Arg
weak interactions for protein binding often come from…
glutamine and adenine
proteins are usually in what form
helix
because can donate R goups
Why are the protein binding interactions to bases called “specific” and those to the sugar phosphate backbone “non-specific”?
backbone is all phosphates: neg charges, so any pos charge can interact with it. same all the way
bases: Specific!
important b/c protein stops at base it wants, and its wobbly, so it binds to the phosphate to lock into place
Lac repressor
protein dimer binds to DNA
causes formation of DNA loop to inhibit transcription
