Lecture 9 Flashcards
What is DNA
the genetic material of all free living organisms
(some ___ – which are not necessarily “living organisms”, have an RNA genome)
Viruses
What is a gene
The part of a DNA molecule that encodes the information required for producing a functional product (-> RNA -> protein)
What is the primary structure
the nucleotide sequence, e.g. :
5’-GCGGCAATCGTA-3’
What is the secondary structure
any regular, stable structure adopted by a segment of DNA, typically a base-paired double helix
What is the tertiary
- the 3D fold - the complex folding of DNA into bacterial nucleoids (supercoiled) or eukaryotic chromatin
- or RNA into tRNA and other molecules
The basic building block of DNA is a nucleotide, which is comprised of:
phosphate + sugar + base
Which way do DNA strands run?
antiparallel directionality of DNA strands… 5’ → 3’
What is on the 1’ carbon? 3’? 5’?
1' = Purine or pyrimidine base 3' = OH group 5' = phosphate group
What does a nucleoside consist of?
base + sugar
The pentose sugar is ___ in RNA and ___ in DNA
Ribose in RNA and deoxyribose in DNA
Name the 2 purine and 3 pyrimidine bases
Purine - Adenine and guanine
Pyrimidine - Thymine and cytosine and uracil (RNA)
In RNA the 2’ carbon has a ___ group?
In RNA the 2’ carbon has a hydroxyl group (2’-OH)
In DNA the 2’ carbon has a ___ group?
a hydrogen
i.e., it has been “deoxygenated”
Is the 2’OH on the RNA or 2’H
The H at the 2’ C of DNA is less reactive than the 2’-OH of RNA,
The pentose in each nucleotide is attached to the base via which carbon
1’ carbon
The phosphate in each nucleotide is attached to the base via which carbon
5’ carbon of the pentose and is thus called the 5’ phosphate (5’-PO4).
The 3’ hydroxyl (3’-OH) of one nucleotide is linked to the ___ of an adjacent nucleotide to form the repeating sugar-phosphate backbone of DNA and RNA.
5’-PO4
The phosphate groups link the pentoses in
both DNA and RNA strands via a
____ bond.
phosphodiester linkage/bond
Why are DNA/RNA strands said to be asymmetric or polar
They have a free 5’-phosphate at one end – the “5’ end” - and a 3’-hydroxyl at the
other end – the “3’ end”.
At physiological pH the acidic phosphate
group of every nucleotide within the
DNA/RNA strand is ___ and thus carries a net ___ charge
At physiological pH the acidic phosphate
group of every nucleotide within the DNA/RNA strand is deprotonated and thus carries a net negative charge
The phosphate groups are acidic or basic?
Acidic
Because of these acidic phosphate groups
(and despite having nitrogenous bases),
DNA and RNA polymers are called
__ acids
Nucleic acids
What are the bases in nucleotides?
- nitrogenous heterocyclic ring structures which include purines and pyrimidines.
- called “bases” because some of their ring nitrogens can be protonated
The pentose forms a covalent bond with the base via a ____ linkage
Beta-glycosidiclinkage
In a b-linkage the base lies ___ the plane of the sugar
How does this differ if it was in alpha-linkage
- above the plane of the sugar
- in alpha, base would lie below the plane, where the H is
What are the Purine deoxyribonucleotides? Give the base name and base + sugar name
- adenine (base) – adenosine (with sugar)
- guanine (base) – guanosine (with sugar)
What are the Pyrimidine deoxyribonucleotides? Give the base name and base + sugar name
- cytosine (base) – cytidine (with sugar)
- thymine (base) – thymidine (with sugar)
Give the nucleotide names for the deoxyribonucleotides
- deoxyadenosine 5’- monophosphate, deoxyguanosine 5’- monophosphate, deoxythymidine 5’- monophosphate, deoxycytidine5’-monophosphate
2 ways ribonucleotides differ from deoxyribonucleotides
- have a hydroxyl at the ribose 2’ carbon
* have uracil base instead of thymine
How do uracil and thymine differ
(uracil is like thymine but without the
methyl group)
What is uridine?
Uracyl + ribose
What are the nucleotide names for ribonucleotides?
- adenosine 5’- monophosphate - guanosine 5’- monophosphate - uridine 5’- monophosphate - cytidine 5’- monophosphate
The nucleotide sequence is written and read from the ___’ to the ___’ end
5’- to the 3’- end
If the nucleotide sequence only has a few AA’s, what is it called?
oligonucleotide
two DNA strands interact via ___ bonds between the
___
two DNA strands interact via hydrogen bonds between the
bases: base-pairing
together the strands form a twisted ladder, with the sugarphosphate backbone and the base pairs forming which parts of the ladder
sugarphosphate backbone forming the sides of the ladder and the base pairs forming the rung
• two strands of DNA are wound around the same
axis to form a ____-handed double helix
Right-handed
What direction do the DNA strands run?
the strands run in opposite directions, one 3’ to 5’ and the other 5’ to 3’: antiparallel
How do the bases line up in terms of the center of the double helix?
the bases are “offset” from the center of the double helix (i.e., if you look end-on, you’ll see that the bases don’t cross through the central axis of the double strand)
___ means hydrogen bonding between the
bases in the two DNA strands
base-pairing
• pairing is specific based on H-bond complementarity: guanine forms ___ H-bonds with cytosine, adenine forms ___ H-bonds with thymine (or uracil in RNA, A=U)
- G-C = 3 H bonds
- A-T = 2 H bonds
• Watson and Crick determined the double helix
structure of DNA that is made possible because of
the specific base pairing
• the specific pairing of bases (G-C, A-T) permits the duplication of genetic information because each strand is a ___ for its complementary strand
template
The G-C interaction, with 3 H-bonds, is ____ than the A-T interaction, with 2 H-bonds.
stronger
In addition to H-bonding between base pairs, the double helix is held together by a second force between the bases: ____
base stacking
Base stacking between the hydrophobic bases
does what to the double helix?
Base stacking between the hydrophobic bases minimizes their contact with water and stabilizes
the double helix.
Base stacking is a form of what force?
van der Waals force
Are bases stacked directly on top of one another?
- bases are slightly offset so they are not directly on
top of one another
What direction do bases lie to the axis of the helix
- the bases actually lie in a plane almost
perpendicular to the axis of the helix
How many base pairs per turn in DNA?
10 base pairs/turn
The major and minor grooves are equivalent or non-equivalent
non-equivalent
Why are the major and minor grooves non-equivalent
Because the two glycosidic bonds are not diametrically opposite each other (i.e., are offset relative to the axis of the double helix), each base pair has a larger side that defines the major groove and a smaller side that defines the minor groove.
The grooves are lined by what bonds
Hydrogen bonds
that functional groups on the bases are more accessible to proteins in the which groove
major groove, rather than minor
• Watson and Crick based their double helix model on ?
the X-ray diffraction pattern of DNA fibers in the B form
What is B form? Where found? Left or right handed? Most stable when?
- B-helix, B-DNA
- In cells
- Right handed
- Most stable under physiological conditions
when does DNA assume A-form?
when DNA is dehydrated (relative humidity is reduced to less than 75%) DNA assumes what form
A form DNA, like B-DNA, is a ___-handed double helix made up of ___ strands held together by ___ base pairing
- RIght handed
- Anti-parallel
- Watson and Crick base-pairing
Which is wider and shorter, A-helix or B-helix
• the A-helix is wider and shorter than the B-helix
Which has deeper major groove and more shallow minor groove, A-helix or B-helix
A-helix
In A-helix are the base pairs tilted or straight
tilted
Where are A-helix frequently found in?
DNA-RNA hybrids
and in double-stranded RNA (e.g., tRNA)
How does Z-form DNA compare to A and B forms?
left-handed and “stretched out compared to A and B forms
Nucleotide bases absorb UV light at __ nm?
260 nm
A260 can be used to determine DNA ___ and ___
Concentration and structure
Since purine and pyrimidines are highly conjugated, what does this allow
resonance among rings give most of the bonds a partial double-bond character, allows UV
absorption (just like for aromatic amino acids)
A260 changes depending on the ____
on the structure of the DNA: single-stranded or double-stranded
double-stranded DNA (dsDNA) helix can be
disrupted by ___, resulting in ____ as the product. After this conversion, ___ and ___ are disrupted
- Heating
- ssDNA
- Base-paring and base stacking are disrupted
What is reanneal? Why does this happen
When the DNA is cooled, the strands
reanneal (come back together) due to base
complementarity: ssDNA -> dsDNA
stacked bases in nucleic acids (i.e., in a double helix) absorb \_\_\_\_ UV light than unstacked bases (i.e. single strands)
Less
What is hypochromisn
absorbance is “quenched” when strands come together
double-stranded DNA is held together by ____ bonds and ____ interactions
- Hydrogen bonds
- Base stacking interacting
DNA can be melted apart by____ or by adding ____
raising the temp or by adding chaotropic (denaturing) agents like urea, and by removing salt
What does highly cooperative mean in terms of DNA?
both the formation and disruption of the DNA double
helix are highly cooperative - the strands hold fast
until the melting point, Tm, and then rapidly let go
What is Tm
melting temp. – point at which strands are ½ denatured
DNA melting (denaturation) and reannealing (renaturation) can be followed by studying the ____
hyperchromatic shift
Melting occurs at a specific Tm depending on what 3 things
the nucleotide sequence,
length, and salt concentration
Why do AT-rich regions melt first?
A=T has only 2 H-bonds so has a weaker interaction than GºC with 3 H-bonds; the more GC pairs, the higher the Tm
Why is Tm is also proportional to [salt] and sequence length
high salt concs and longer sequences stabilize the duplex and increase the Tm. Salt ions shield the negatively-charged phosphates on the DNA backbones, which can repel
each other when unshielded.
So, how do cells contain/package/handle their DNA?
Bacterial DNA can be compacted by a process called supercoiling into a nucleoi. DNA is condensed, organized and segregated with the help of topoisomerase enzymes, nucleoid associated proteins and Structural Maintenance of Chromosome (SMC) complex
Supercoiling resuts from slighty ___ or ____ the DNA double-helix prior to joining both ends of each strand. Enzymes called ____ perform this task
- Overwinding (DNA can only be supercoiled by breaking the strands,
twisting them around each other and religating them) or under winding - Topoisomerases
Supercoiling can only exists in what kinds of molecules
Supercoiling can only exist in a molecule where both strands of DNA are closed circles or otherwise fixed at one end
What happens when one stand breaks?
If one strands breaks the DNA rapidly loses its supercoiling
i.e., it relaxes
DNA molecules in different coiled forms are called?
Topoisomers
DNA supercoiling helps to compact DNA but it is also a consequence
of _____
DNA unwinding during replication and transcription (RNA synthesis)
Most cellular DNA is overwound or underwound?
Underwound, - DNA has fewer helical turns than would be expected for the B form
relaxed DNA has ___ base pairs per turn
~ 10.5
a segment of DNA in a closed circular
molecule is relaxed with ___ bases in
8 helical turns
84
removal of one turn (unwinding) induces ___
structural strain
To accommodate the strain from removal of one turn (underwinding), the DNA can either ___ or _____
- become supercoiled
- or break H bonds
Why is the strain generally accommodated by formation of supercoil rather than breaking H bonds
It disperses the
strain throughout the molecule. This requires less energy than breaking the H bonds that
stabilize paired bases.
Cells actively underwind their DNA using ____, and the resulting strained state represents a form of ___ energy
- Topoisomerases
- Stored energy
The underwound state can be maintained only if?
the DNA is a closed circle or if it is bound and stabilized by proteins so that the strands are not free to rotate about each other
This underwound, supercoiled state
allows for what of the DNA?
This underwound, supercoiled state
allows compact packaging of the DNA
What is Lk
- linking number
- the number of times one strand crosses
over the other.
Does the Lk change when DNA is bent or deformed?
No as long as both DNA strands
remain intact. i.e., it is a topological
property of the DNA
How can Lk be changed
Lk can only be changed by breaking one or both strands and re-joining (re-ligating) them.
Are Lk for a closed-circular DNA always an integer?
Yes
By convention, if the links between two DNA strands are arranged so that the strands are interwound in a right-handed helix, Lk is + or -. Linking numbers in a left-handed helix are + or -?
- Right-handed helix = pos
- Left handed helix = neg
The double helix is a right-handed helix so cellular DNA has a +/- linking number?
+
The interwinding of the supercoils can be ____ (underwound, right-handed helix) or ____ (overwound, left-handed helix)
- Negative = underwound, right-helix
- Positive = overwound, left helix
For relaxed DNA, Lk = ?
- # of base pairs divided bythe # of base pairs per helical turn in relaxed DNA
(i.e., it is equal to the number of turns). - So for a circular
piece of relaxed DNA with 2100 base pairs,
Lk = 2100 bp/10.5 bp per turn = 200 (turns)
Does Underwinding or overwinding changes the linking number?
Yes
For the relaxed DNA molecule,
underwinding or overwinding by two helical turns (i.e., to Lk = 198 or 202) will produce what kind of supercoiling
- Underwinding = negative supercoiling
- Overwinding = positive supercoiling
Negative supercoiling results in a ___ handed helix whereas positive supercoiling is ___-handed.
- Negative supercoiling results in righthanded helix
- Positive supercoiling results in lefthanded helix
Can relaxed or supercoiled DNA lie flat on a planar surface
- Supercoiled
Why is supercoiled DNA called the tertiary structure?
Supercoiled DNA represents higher
order folding of secondary structure, or helix
A supercoiled DNA molecule is more or less compact than a relaxed DNA molecule
of the same length
More
Supercoiling allows small bacterial cells
to ____ long lengths of DNA
Package
Supercoiled DNA is more compact: it moves ___ than relaxed DNA when subjected to
centrifugation or gel electrophoresis.
Faster
The only way to change Lk for a circular molecule is to ?
cut it then twist and rejoin the ends
topoisomerases are enzymes that play an especially important role in processes such as
Replication and DNA packaging
What are Topoisomerases
enzymes that catalyze interconversion of topoisomers (changes superhelicity)
Topoisomerases change the
linking number of DNA by catalyzing what three-step process
1) the cleavage of one or both strands of double-stranded DNA
2) the passage of a segment of
DNA through this break
3) resealing of the DNA breaks
How do topoisomerases play important roles
in replication, transcription and
recombination for both prokaryotes
and eukaryotes
help relieve
torsional stress induced by unwinding the DNA during these events
How do topoisomerases utilize the key tyrosine residue
Topoisomerases utilize a key tyrosine residue that covalently attaches to a phosphate in the sugar-phosphate backbone that is transiently broken.
Describe Topo I (4)
- nicks one strand of DNA - the other strand then passes through the ss break, and the broken strand re-seals
- changes the linking number (DLk) by increments of 1
- thermodynamically favorable process driven by release of energy of supercoiling
- E. coli type I topoisomerases generally relax DNA by removing negative supercoils (increasing Lk)
How was topo II (DNA gyrase in bacteria)
- breaks both strands, and then re-seals both strands • DLk in increments of 2
- can relax negative supercoils
- in E. coli, Topo II (DNA gyrase) can also introduce negative supercoils using energy from ATP hydrolysis (decrease Lk)
The degree of supercoiling of DNA is maintained by regulation of the net activity of
topoisomerase types I and II
Topoisomerases are the molecular targets of _____ and _____
antibacterial and anti-cancer drugs
Without topoisomerases, cells
cannot ___ or ____ or ____
replicate or package their DNA, or express their genes so they die
Topoisomerases have therefore
become important targets for inhibitors
Pharmaceutical agents that specifically inhibit ___ are effective in treating infectious bacteria and malignant cells
topoisomerases
The quinolones inhibit?
bacterial DNA gyrase, a Type II topoisomerase.
The fluoroquinolone Ciprofloxacin (Cipro) is one of the few antibiotics effective in treating?
anthrax infections; useful for other bacterial infections as well
How and why do anticancer drugs target topoisermerases
Anticancer drugs also target topoisomerases, which are elevated in tumor cells. Agents targeted to these enzymes are thus more toxic to tumor cells than to normal cells. They act by inhibiting Topo I or Topo II.
Like proteins, DNA fragments can be separated based on their size using ___
Electrophoresis
DNA fragment migration in electrophoresis depends on?
Their degree of supercoiling
Why is DNA naturally negatively charged?
Because of the negatively-charged phosphates in the sugar-phosphate backbone
Which section will DNA migrate to in electrophoresis in an buffered agarose gel
Anode (+)
Do you need to add SDS during electrophoresis for DNA
No
Do smaller or larger fragments migrate more quickly?
Smaller
The DNA bands can be visualized how?
Under UV light by staining with ethidium bromide
What is ethidium bromide
a dye that binds the DNA by intercalating between the bases
Why do supercoiled DNA runs more quickly than relaxed DNA
Because it is more compact
How does treatment with Topo I change DNA?
All DNA circles run on this agarose gel are the same length but differ in Lk after treatment with Topo I. DNA in lane 3 has been treated with Topo I for longer than that in lane 2. With long enough Topo I treatment, all DNA circles will eventually become relaxed.
each base pair is more exposed to solvent on which groove more so
Major groove
on either side (especially the major groove side) there is potential for the base pairs to H-bond with what?
compounds in the surrounding environment, like water or proteins
fxnal groups of base pairs that can be accessed from grooves: “exocyclic groups” -NH2, -C=O; ring N – these groups are not involved in base pairing and are exposed in the grooves… what does this allow
serve as contact points for proteins searching for specific sequences on the DNA – recognize a DNA sequence based on the base pair functional groups exposed in the groove
