bioch lec 2
what was the end result of the avery macleod mccarty experiment
transforming principle is heat killed smoth strain trasnformed rough strain to s strain and killed mouse
T2 Phage: dna is high in, protein coat is high in
dna is high in P where protein coat is high in s
what did the hershey chase experiment show (1952)
after infection, blending and centrifugation what stayed behind in the progeny is the phosphorous (radioactivie)--> the dna stayed.
what are roles of polynucleotides
-storage and decoding genetic information
- enzymes
a nucleotide is made of three parts:
1. nitrogenous bases
2. sugar (ribose or deoxy)
3. phosphate groups
when nucleotides are joined by phosphodiester bonds they can form.... how?
DNA or RNA. phosphate of one nucleotide bonds to the 3 'OH of the sugar in another nucleotide( sugar phosphate backbone)
what else can nucleotides do besides forming dna and rna.
can remain as high energy molcules: ATP and GTP (NTPS)
ex. atp has 3 phosphate groups linked by phosphoanhydride bonds which are unstable bc the negative charges on phosphate repel each other (bond strain) when broken relieves this repulsion and releases energy.
can also be modified to carry and transfer electrons during redox (NADH, FADH2, FMNH2, NADPH)
what are the 5 common bases and 2 common sugars
adenine, cytosine, guanine, thymine, uracil
deoxyribose and ribose
draw the 4 common bases (purine vs pyrimidine)
did u do it
what is resonance- can lone pairs in resonance accept hydrogen bonds
resonance occurs when electrons can flow through neighbouring pi (doouble) bonds- delocalize. these lone pairs cannot accept hydrogen bonds
how do thymine and uracil differ
in terms of hydrogen bonding they are the same the only difference is a methyl group on c5 in thymine
which nitrogen links the base to the sugar in pyrimidines vs purines
pyrimidines: N1
Purine: N9
which carbon is different on deoxyribose
carbon 2
what carbons are chiral in ribose? what about deoxyribose ?
ribose: atoms C1-C4
deoxyribose: C5 and C2 are NOT chiral
nucleoside vs nucleotide
nucleoside is only base and sugar
how to name purines vs pyrimidines (nucleosides)
purines: ine --> becomes osine. if deoxy sugar add deoxy at start
ex. adenosine, deoxyadenosine
pyrimidnes: end in idine. if deoxy sugar add deoxy at start. ex. cytidine, deoxycytidine
where do phosphates typically attach in a nucleotide? where can they also attach.
usally c5 (particulary in NTPs), can also attach at c2, c3
an adenine attached to a deoxyribose with 3 phosphates at carbon 5 would be named what?
deoxyadenosine 5' triphosphate (dATP)
if there happens to be thymine in a rare rna molecule what is it called
ribothymidine 5' monophsophate (rTMP)
what is deoxyuridine
a short lived dna intermediate- the deoxy form of uracil used to make thymine during dna synthesis
what is the general structure of nucleic acids
nucleic acids are polymers of nucleotides linked by 3'-5' phosphodiester bonds
phosphoester vs phosphodiester
phosphoesters link carbons to phopshate groups where as a phosphodiester links two different carbon atoms to one phosphate group
what is a phosphoanhydride
links two phosphates to each other
what is the convention of writing nucleotide sequences
they are written from 5' left to 3' right
what creates the asymmetric structure and sense of direction for nucleic acids
phosphodiester bonds
what is the primary structure for a nucleic acid
the exact order of nucleotide residues- the sequence of the bases joined by phosphodiester bonds from 5' to 3'
what is the net charge of dna and rna
negatively charged moclules at physiological pH bc each nucleotide in the backbone has a phosphate group and a t neutral pH the phosphates are deprotonated (have a negative charge)
what is the polarity of the rna and dna backbones
the backbones of both are made of alternating sugar and phosphate groups linked by phosodiester bonds.
phosphate groups: polar and - charge
sugar: polar due to OH
Rna is more polar and less stable because of extra OH on its sugar
when working with dna system, mixed system, rna system what are the conventions
for just dna or rna, optional to classify d prefix or direction
when working witha mixed ysstem classify direction and d prefix or d prefix with (dna) at the end
what does phosphodiesterase do
it is an enzyme that hydrolyzes phosphodiester bond (breaking a bond by adding water), it cuts one o f the ester linkages to produce a 5' phosphate and a 3'Oh + H+ from water
why does RNA undergo spontaneous alkaline hydrolysis and why is DNA stable under the same conditions?
RNA's 2-OH group attacks the adjacent phosphate in basic conditions causing self clevage of the phosphodiester bond. dna lacks this 2'-OH so no nucleophilic attacks occurs making DNA alkali stable
which oh grop acts as the leaving group most likely in the alkaline cleavge of RNA
first the 2'OH is deprotonated and attacks the phosphorous, now the bond between the phosphorous and one of the ozygens must break to release this strain. usually the 3'O-P breaks (major favoured pathway--> forms a stable cyclic phosphate)--> produces a 2'3' cylic phosphate intermediate and 5'OH fragment. after this hydrolsis can can open the ring at either 2' or 3' position
what spontaneous reaction converts cytosine into uracil in DNA
Cytosine deamination loss of an amino group (NH2) (from added water). its a spontaneous uncatalyzed reaction that happens naturally over time.
why is cytosine deamination dnagerous in DNA if left uncorrected
because uracil pairs with adenine instead of guanine. so if the uracil remains during replication the G-C base pair becomes an A-T base pair- a point mutation (transition)
why doess DNA contain thymine instead of uracil
so that uracil generated by cytosine deamination can be recognized as an error. if DNA used uracil normally repair enzymes couldnt tell which U's are real and which came from cytosine damage . Thymine (a methylated uracil) makes it easy to spot and fix deamination errors
what are the main structual and chemical properties of pyrimidines
single ring, heterocyclic, aromatic compounds.
planar and electron delocalized (pi system over the ring)
nitrogens and carbonyl groups make them capable of h bonding
found in c u t
what are the main structural and chemical properties of purines
double ring system
aromatic with electron delocalization over both rings
mostly planar but can have a slight pucker due to steric strain
largely hydrophobic but contain some polar groups (N and O) that allow h bonding
A and G
how do nitrogenous bases interact with water and each other
poorly soluble in water (hydrophobic aromatic rings)
can form hydrogen bonds through N and O atoms (important for base pairing)
stacking interaction (pi-pi interaction) between aromatic rings stabilize DNA and RNA structure
what is more soluble pyrimidine or purines
pyrimidines are slightly better since they are smaller
why do dna and rna absorb uv light
because their nitrogenous bases are aromatic with delocalized pi electrons that absorb strongly at 260 nm
at what wavelenghts do nucleic acids and proteins absorb UV light most strongly
DNA/RNA: max at 260 nm (typical A260/A280 ratio for pure dna 1.95, RNA= 2.0)
proteins: max at 280 due to aromatic amino acids
if a dna sample is contaiminated with protein what happens to the A260/ A 280 ratio
the ratio decreases because
dna absorbs stronly at 260 nm
protein absorb strongly at 280 nm
so contamination adds more 280 nm absorbance which lower the ratio
280 increases in the fraction so the ratio decreases
what is beer lambert law and what does it describe
a solutions absorance to its concerntain and path length. absorbance is directly proportional to both the concentration of the absorbing species and the path length.
phosphoester vs phosphodiester
a phosphoester bond joins phosphate to sugar while phosphodiester bonds join two sugars via phosphate in nucliec acids (3' carbon to the 5' carbon.)
how are the chains in dna connected
through hydrogen bonds between the bases (non covalent)
what did erwin chargaff sugget and who did this help
A=T, G=C, purines=pyrimidines. helped watson and crick deuce the double helical structure of DNA
why must hydrogen bonds between or within biochemical macromolecules be shielded from water
because water itself forms strong hydrogen bonds, it can compete with and disrupt hydrogen bonds inside or between macromolecules. to stay stable intramoclule or intermolecular h bonds must be shielded from water usually by being buried in hydrophobic regions.
the primary structure of nucleic acids is said to have a sense of direction what does this term describe
the free ends of a nucleic acid are structurally and chemically distinct from one another
what allows for bonding interactions between the bases
a-T: can from 2 hydrogen bonds, A's N-H with T's O and A's N with T's N-H
G-c: 3 hydrogen bonds, G's O and N-H's and C's N-H, N and O.
describe dna (b form)
stablized by base stracking and hydrogen bonds.
base stacking (primary stabilsing force): mostly vanderwaals and also hydrophobic forces
strands are antiparallel with an overall right handed twist
ribose.deoxyribose and phosphates exposted to h20
hydrophobic core/polar exterior
bases largely excluded from h20
10 bp per turn
what would the axial view of dna show
the polar sugar/phosphate backbone is found on the outside while hte more hydrophobic bases stack in the core of the structure
what is required for base pair froamtion in dna and what happen to other h bonding groups on the bases
bp requires specfic h bonding between complementary bases on the two strands. the remaining h bonding groups on the bases that are not used in pairing are exposed to the major and minor grooves of the double helix where they can interact with water, ions or proteins (dna protein recognition/solvent interaction)
why do nucleic acids have a sense of direction
direction comes from the asymmetry of the sugar phosphate backbone 5'phospahte to 3' hydroxyl
if a 28 bp segement of double stranded dna contain 7 a residues how many g residues are there
28 bp --> 56 bases
a=t --> 14
g=c 56-14= 42/2= 21
how does dna denaturation occur
seperation of strands, and the breaking of hydrogen bonds between complementary bases without breaking covalent bonds in the backbone happens when heat, high pH or chemical agens disrupt these bonds and base stacking interactiosn that stabilize the double helix
why does dna denaturation happen- is it necessary
denaturation happens when conditions overcome the stabilizing forces of base pairing. its reversible and biologically necessary in processes like replication and transcription where strands must separate to serve as templates for new synthesis
what is dna melting temperature (Tm)? when is it higher
Tm is the temp at which half of the DNA is single stranded it depends on base compositon (midpoint of melting)
higher Tm with higher GC content (since more h bonds/stacking interactions)
this is asigmoidal relationship, cooperative process, and the temp range is relatively small
what happens to UV absoprtion during DNA denaturation
absorbance at 260 nm increases this is called the hyperchromic effect. unstacking/broken hydrogen bonds--> pi electrons more exposed--> greater absorption of uv light
why can dna melting curves be measured
because single stranded dna has a higher absorance than double stranded
hyperchromicity vs hypochromicity
hyper--> relatively high absorbance (SS DNA)
hypo--> low absorbance (DS DNA)
what happens during denaturation and renaturation of double stranded dna
denaturation: heat or high ph break h bonds--> seperate
renaturation (reannealing): cooling or neutralization allows complementary strands to realign and reform base pairs
what are the 2 main steps of dna renaturation (20-25 celsisus below Tm)
1. nucleation (slow): random complementary sequences find eachother and form a small region of correct base pairing
2. zippering (fast): once a short double stranded region forms, the rest of the strand rapidly "zips up" through succesive base pairing
what factors affect the rate of dsDNA renaturation
sequence complexity: simple (repetitive) sequences reanneal faster; complex genomes reanneal slower
temperature: optimal renaturation occurs just below the melting temp (25c below tm), too low slows diffusion, too high prevents pairing
concentration: higher dna conc increases collision frequency--> faster nucleationg
what has higher tm, at pair or gc pairs
GC pairs have an elevated Tm as they have stronger base stacking interactions
what type of duplex dna sample would have the highest tm
one with the highest gc content or the lowest at content
how does ph affect tm
extreme ph (high or low)--> lowers tm
high ph (alkaline): deprotonates bases--> breaks hydrogen bonds
low ph (acidic) protonates bases--> disrupts base pairing
both conditons destablize the double helix causing dentauration at lower temps
how does salt conc effect tm
increasing salt conc (Na+, K+)--> increases Tm--> stabilizes the double helix. postive ions (mg2+/ postive proteins-histones) shield the negative charges on the phosphate backbone reducing electrostatic repulsion between strands
low salt or no salt--> decreases tm bc repuslion between phosphate groups makes the strands easier to seperate
how does rna form different structures than dna
1. A pairs with U
2. often single stranded
3. intrastrand (within one strand) base pairing--> creates loops, hairpins, complex 3d shapes, making rnas structures highly dependent on its nucleotide sequence
vs dna which is interstrand--> forms a stable double helix that is relatively independent of sequence
if you wanted to favour hybridization between only partially complementary (imperfectly matched strands can hybridize-low stringency) sequences which of the following conditions would you choose
use high salt conc-> higher tm/stabilize imperfect duplex
can also do low temp, second option, less energy available to break mismatched bps
what type of base pairs stabilize complex rna secondary structures
rna folds into complex secondary structures (hairpins, loops, bulges) stabilized by:
w-c pairs:
G=C (3 h bonds)
A=U (2 h bonds)
non w-c pairs:
(wobble) G=U: forms 2 h bonds, slightly weaker but stable
sequence-->structure (spontaneous)-->function
is the dna and complementary RNA double stranded helix the same structure as B-DNA ? how does it pair?
no
A=U
T=A
G=C
antiparallel also, right handed also, base stacking interactions also
rna does not melt because its single stranded
false, partially ds so can melt but differently than dna
the 3d structure of rna is more stable than that of dna
stability not signifcantly different
how do you read the complement strand
3' to 5'