MBB 3

DNA to Protein

DNA Synthesis - Replication

RNA Synthesis - Transcription

Protein Synthesis - Translation

Amino Acids

DNA primary structure

nucleotide

• phosphate

• sugar

• base (purine or pyramidine)

Pentose Sugar - RNA

Ribose

Pentose Sugar - DNA

deoxyribose

Difference of structure in DNA and RNA

RNA the 2’carbon has a hydroxyl group while in DNA the 2’ carbon has a hydrogen group

What is relevant in RNA splicing

2’-oh

Pentose in each nucleotide is attached to the base via?. The phosphate is attached to the ? carbon is called the ?

Pentose in each nucleotide is attached to the base via 1’carbon. The phosphate is attached to the 5’ carbon is called the 5’phosphate

What 2 parts of nucleotides are linked to crease the repeating sugar phosphate backbone

2’Hydroxyl and 5’PO4

Nitrogenous heterocyclic ring structures

Purines and Pyrimidines

• highly conjugated aromatic rings

Why are bases called bases

because some of their ring nitrogen can be protonated

Purines

Adenine

Guanine

Pyrimidines

Cytosine

Uracil

Thymine

Where does pentose attach on Purines

On C9

Where does pentose attach on Pyrimidines

C1

Pentose forms a covalent bond with the bases via a B-glycosidic linkage

a B-glycosidic linkage

Why not form covalent bonds with a-linkages

the base would lie below the plane where the H is

How to distinguish A from G

Guanine has a double bonded oxygen sticking up

How to distinguish T from C

Thymine has 2 double bonded O groups while Cytosine has 1

What is the difference between deoxyribonucleotides and ribonucleotides

What is the difference between U and T

uracil does not have a methyl group (CH3)

Nucleotide Sequence

5’-3’

pACGTA is an example of how one would write a sequence

What tells you is its a 3’ or 5’ end

5’ has a free phosphate group

3’ has a free OH group

If it only has a few residues (nucleotides) it is a

an oligonucleotide

The phosphate group link the pentoses via

phosphodiester bonds

The strands are said to be

polar

The phosphate group is an acid or base what is the impact

Acid. At the physiological pH the phosphate group of every nucleotide within the strand is deprotanated so carries a net charge of -1

Why does the phosphate group carry a net charge of -1

This occurs because each internal phosphate is linked in a phosphodiester bond involving two of its oxygen atoms, leaving one remaining acidic hydroxyl group that is fully deprotonated at cellular pH levels

• Each linked internal phosphate carries a single negative charge (-1) due to the ionized oxygen

• This repeating negative charge gives the entire sugar-phosphate backbone of DNA and RNA its strong overall negative character

Why the name nucleic acids

because of the acidic phosphate groups

Two DNA strands interact via

hydrogen bonds

A has 3 bonds with T/U

G has 2 bonds with C

Who proposed base pairing specificity and based on what

Watson-Crick base pairing based on x-ray diffraction data

The specific pairing of bases permits ?

the duplication of genetic info because each strand is a template for its complimentary strand

In addition to H-bonding between base pairs, the

double helix is held together by

base stacking

Base Stacking

between the hydrophobic bases minimizes their contact with water and stabilizes the double helix

• base-stacking is a form of van der Waals forces

• bases are slightly offset so they are not directlynon top of one another

• the bases lie in a plane almost perpendicular tomthe axis of the helix

the offset pairing of the two strands (i.e., bases not in the center of the double strand) forms

a major groove and a minor groove on the surface of the duplex

• grooves lie on opposite faces of the double helix and twist around the helix axis

• so that if you see a major groove facing you, the minor groove is on its back side

The base pairs are more exposed to solvent on the _______ groove side than on the ______ groove side. Why?

The base pairs are more exposed to solvent on the major than on the minor groove side.

Major is more spacious so water and protein easier access to base edges

Minor is narrow so limits direct access

The forms of the double helix

1. B-DNA

2. A-DNA

3. Z-DNA

B-DNA

most common

right handed helix

10.5 pairs per complete turn with wide major and narrow minor

A-DNA

occurs in dehydrating conditions - standards for double stranded RNA or hybrids

• right handed but shorter and wider than B

  • 10.7 pair per turn with bases tilted away from central

Z-DNA

high energy conformation forming under high salt or supercoiling stress

• left handed helix with a zigzag backbone

• narrower and elongated 12 base pairs per turn

Label the DNA

A

B

Z

Nucleotide bases absorb UV light at? and can determine?

260nm

DNA concentration

What allows UV absorption in nucleotides

purines and pyrimidines are highly conjugated - resonance among rings give most of the bonds a partial double-bond character, allows UV absorption

Beers Law

the amount of light absorbed by a solution is directly proportional to its concentration and the distance the light travels through it

Beers law formula

A = ε ⋅ l ⋅ c [

• A: The absorbance of the solution (which is unitless).

• ε (epsilon): The molar absorptivity (or molar extinction coefficient). It measures how well the chemical species absorbs a specific wavelength of light (units are usually L ⋅ mol⁻¹ ⋅ cm⁻¹).

• l: The path length, which is the distance the light travels through the solution (usually in centimeters).

• c: The concentration of the absorbing substance in the solution (usually in molarity, mol/L).

A260 can also be used to distinguish between single-stranded and double-stranded DNA

• double-stranded DNA (dsDNA) helix can be disrupted by heating

• The melting point is determined by nucleotide sequence

• Once cooled the strands re-anneal - come back due to base complementarity

• The stacked bases absorb less UV that unstacked so absorbance is quenched when strands come together

DNA melting (denaturation) and re-annealing (renaturation) can be followed by studying the ?

hyperchromic shift

hyperchromic shift

the large increase in ultraviolet (UV) light absorption by nucleic acids when double-stranded DNA or RNA unwinds into single strands. It happens because unstacked bases in single strands absorb more light than stacked bases in a double helix

• absorption intensity e is increased

• shift due to change in structure

• UV absorption at 260 nm spikes by about 37%

Why DNA Denaturation Causes the hyperchromic shift

Since the nitrogenous bases are stacked tightly on top of each other it limits their ability to absorb UV light

• therefore when heat or chemicals denature the strands the bases get fully exposed to light

• this allows the bases to absorb more UV light causing the shift

Tm

the temperature at which

half the DNA is in a ds form, half is in a ss

form.

Melting occurs at a specific Tm depending on

nucleotide sequence

length

concentration of salt in solution

The stacking energy is more negative (more stable) for ? pairs, so Tm is higher. The ? regions melt first.

The stacking energy is more negative (more stable) for GC pairs, so Tm is higher. The AT regions melt first.

Tm is also proportional to [salt] and sequence length

high salt concentrations and longer sequences stabilize the duplex and increase the Tm

• More sodium or magnesium ions neutralize the negative charges of the DNA strands, which reduces the pushing force between them and helps them stick together tighter.

• Longer DNA strands have more hydrogen bonds and base-stacking forces holding the two strands together, which requires more heat to pull them apart.

way of compacting DNA

DNA superhelicity

How do cells contain/package/handle their DNA?

• Bacterial DNA can be compacted by a process called supercoiling into a nucleoid.

• DNA is condensed, organized and segregated with the help of topoisomerase enzymes, nucleoid associated proteins and the Structural Maintenance of Chromosome (SMC) complex.

A closed double-stranded molecule of DNA can be compacted by

supercoiling

Supercoiling

can only exist in a DNA molecule where both strands of DNA are closed circles or otherwise fixed at one end. If one strands breaks the DNA rapidly loses its supercoiling

DNA molecules in different coiled forms that have the same nucleotide sequence are called

topoisomers

Supercoiling

The topology (shape) of dsDNA can be defined in terms of

Linking number

Linking Number

the number of times one strand

would have to be passed

through the other strand in

order for the strands to be

completely separated from each

other

Linking number formula

Lk=Tw+Wr

Tw

• Twist

• For dsDNA, twist is the number of full turns of the helix

one turn of a B form helix is 10.5 bps, Tw of a

segment of this relaxed DNA which is 105 bp long

will be:

Tw=105/10.5 = 10

Wr

• writhe

• measure of the degree of supercoiling

• The number of times double helix crosses itself

if ds helix writhes in the left- handed direction

Wr is assigned a positive value

• Overwinding (+) makes it more difficult to separate the strands of the double helix

If helix writhes in the right-handed direction

Wr is assigned a negative value

• Underwinding (-) makes it easier to separate the strands of the double helix

Wr for relaxed dsDNA

0

Lk

a topological property of the circular DNA - it cannot be changed unless one or both of the strands of the duplex is broken

What moves faster in centrifugation or gel electrolysis supercoiled or relaxed DNA and why

A supercoiled DNA molecule is

more compact than a relaxed

DNA molecule of the same length:

it moves faster than relaxed DNA

when subjected to centrifugation or

gel electrophoresis.

What kind of structure is supercoiled DNA

tertiary structure

DNA gel electrophoresis

The migration also depends on their degree of supercoiling

• Since DNA has a net - charge it will migrate towards the anode (+) in agarose gel.

• Smaller DNA move faster through than less supercoiled on same size

• More supercoiled fragments move faster than less supercoiled of the same size

The DNA bands can be visualized under UV light by staining with

ethidium bromide

ethidium bromide

a dye that binds the DNA by intercalating between the bases

DNA gel electrophoresis

Separating DNA fragments based on their sizes (larger

or smaller) and shapes (supercoiled or relaxed)

Supercoiled the fastest and furthest

Linear depends on true size in base pairs

Relaxed migrates the slowest and shortest distance

Topoisomerases change the linking number of DNA by catalyzing a 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

Topoisomerases have a key

tyrosine residue in their active site that covalently attaches to a phosphate in the sugar-phosphate backbone that is transiently broken

Type I topoisomerase

• Type I topoisomerase is an enzyme that relaxes DNA strands by cutting a single strand, passing another strand through the break, and resealing it.

• No ATP

• Changes DNA linking number by 1

• Type 1 relaxes supercoiled DNA by letting one strand rotate around the other

• Thermodynamically favorable process driven by release of energy of supercoiling

• relax DNA by removing negative

  supercoils (increasing Lk)

Type II topoisomerase

• cuts both strands of the DNA double helix

• needs ATP

• DNA linkages by 2

• untangle, unknot, and separate linked rings of DNA (decatenation) in addition to relaxing supercoils.

Type I mechanism

• Cleavage:

  • Uses a tyrosine residue to attack and break one phosphodiester bond on a single DNA strand.

• Passage:

  • The intact strand passes through the nick, or the cut strand rotates around the uncut strand.

• Religation:

  • The enzyme seals the broken strand back together without consuming external energy

Type II Mechanism

• Binding:

  • Grabs onto one DNA segment (the G-segment) and binds ATP.

• Cleavage:

  • Breaks both strands of the G-segment, forming a transient protein-gate.

• Passage:

  • Passes a second, intact DNA duplex (the T-segment) completely through the gate.

• Religation:

  • Reseals both strands of the G-segment using energy from ATP hydrolysis.

Topoisomerases are the molecular targets of

antibacterial and anti-cancer drugs

The topological state of cellular DNA is intimately connected with its function

Without topoisomerases, cells cannot replicate or package their DNA, or express their genes, so they die

The quinolones inhibit bacterial DNA gyrase, a type II topoisomerase.

Quinolones act by blocking the last step of the topoisomerase reaction, the resealing of the DNA strand breaks. This action inhibits the unwinding of the bacterial chromosomal DNA during and after the replication, thus preventing bacterial cell division.

Three hypothesized mechanisms for DNA replication

The Meselson–Stahl experiment

provided biochemical evidence

that DNA replication is

semiconservative.

How was this experiment done?

semi-conservative replication using nitrogen isotopes and density gradient centrifugation. coli were first grown in heavy nitrogen (¹⁵N) medium. They were then shifted to light nitrogen (¹⁴N) medium. DNA density was analyzed using cesium chloride gradient centrifugation.

The resulting DNA bands proved that each new double helix consists of one original parent strand and one newly synthesized strand

What are DNA building blocks?

nucleotides

How many phosphates do dNTPs have?

3

alpha

beta

gamma

starting from the one closest to sugar

Primer to provide what

OH on 3’

DNA polymerase is an

enzyme

Required cofactors for DNA replication

Mg2+ to facilitate nucleophilic attack

The raw materials for DNA synthesis/replication are:

1. a DNA templatedeoxynucleoside triphosphates (dNTPs)

2. a DNA or RNA ‘primer’ to provide the first 3’-OH

3. Mg2+ ions (cofactor for thepolymerase)

4. DNA polymerase - a large protein complex that includes the enzyme that catalyzes the addition of dNTPs on the 3’ OH end of the primer

The DNA polymerase reaction adds a

deoxynucleotide to the 3' end of thegrowing DNA chain.

The Mg2+ ions

stabilize the negative charges on the

deoxynucleotide and assist in deprotonation of the 3'-OH by a base

The 3'-oxygen of the growing DNA strand serves

as the nucleophile in this reaction, displacing the pyrophosphate from the

deoxynucleoside triphosphate (dNTP) in the active site. The product is a DNA

strand that has been extended by one nucleotide in the 3' direction.

The shape of the DNA polymerase active site facilitates

formation of the correct Watson–Crick base pairs: correct bases pairs fit well into the DNA polymerase active site, mismatches do not

• high fidelity

• base selection

  • steric clashes

What is an endonuclease?

an enzyme that cleaves the internal phosphodiester bonds within a polynucleotide chain (DNA or RNA)

What is an exonuclease?

type of enzyme that degrades nucleic acids (DNA or RNA) by removing nucleotides one by one from the ends of the strand, rather than cutting them in the middle

What is a 5’-->3’ exonuclease?

A 5’-->3’ exonuclease is an enzyme that removes nucleotides one by one from the 5' end of a DNA or RNA strand, moving toward the 3' end

What is a 3’à5’ exonuclease?

A 3’→5’ exonuclease is an enzyme that removes DNA or RNA building blocks (nucleotides) one by one from the 3' end of a nucleic acid chain

DNA synthesis occurs at

replication forks simultaneously for both parent strands, always in the 5’→3’ direction.

DNA is partially unwound at “replication forks.” Both strands are used as templates for the

synthesis of new strands in a 5’→ 3’ direction.

• The leading strand is synthesized continuously

• the lagging strand is, by necessity, is synthesized in short pieces termed Okazaki fragments (100s to 1000s of nucleotides).

• Both new strands are synthesized in a coordinated fashion by a single multimeric DNA polymerase III complex (DNA pol III).