Lecture 6

Lecture 6Nucleotides and Nucleic Acids

Clarification for the previous lessons 2,3-disphosphoglycerate (2,3-DPG ) = 2,

3-bisphosphoglycerate (2,3-BPG) Hemoglobin saturation curve =

oxygen–hemoglobin dissociation curve

oxygen–hemoglobin dissociation curve

CO2 ?

Sample question

The level of carbon dioxide in the blood affects the oxygen carrying capacity of hemoglobin in two ways. Describe the dual effect of CO2 on Hb.

Hints: (1) H2O + CO2 H2CO3 H+ + HCO3- ; alter blood pH

(the Bohr Effect); (2) Hb·NH2+CO2 Hb·NH·COOH ; carbamino

Generally, CO2 pressure increase curve right shift (Low oxygen binding affinity)

Other factors interfering with O2 loading: Carbon monoxide - displaces oxygen from hemoglobin Methemoglobinemia Fe2+ → Fe3+  (doesn't combine with O2)

Sample question

What is the shape of the oxygen hemoglobin dissociation curve?

How does the shape of the curve relate to the cooperative binding of O2?

How does its shape influence loading of oxygen at the lung and unloading of oxygen at the tissue level?

What causes oxygen movement into and out of the blood?

Information Transfer in Cells

Information encoded in a DNA molecule is transcribed via synthesis of an RNA molecule

The sequence of the RNA molecule is "read" and is translated into the sequence of amino acids in a protein.

Nucleic Acids

Compound contained C, N, O, and high amount of P.

Was an acid compound found in nuclei therefore named nucleic acid

Nucleic Acids

Nucleic acids are long polymers of nucleotides.

Nucleotides contain a 5 carbon sugar, a weakly basic nitrogenous compound (base), one or more phosphate groups.

Nucleosides are similar to nucleotides but have no phosphate groups.

Nitrogenous Bases

Pyrimidines Cytosine (DNA, RNA) Uracil (RNA) Thymine (DNA)

Purines Adenine (DNA, RNA) Guanine (DNA, RNA)

Nitrogenous Bases

Properties of Pyrimidines and Purines Keto-enol tautomerism

Strong absorbance of UV light

absorbance of UV light

Pentoses of Nucleotides

D-ribose (in RNA) 2-deoxy-D-ribose (in DNA) The difference - 2'-OH vs 2'-H This difference affects secondary structure

and stability

 L-ribose and L-deoxyribose not found in nature

D-amino acids is rare.

NucleosidesLinkage of a base to a sugar

Base is linked via a glycosidic bond The carbon of the glycosidic bond is anomeric Named by adding -idine to the root name of a

pyrimidine or -osine to the root name of a purine

Conformation can be syn or anti Sugars make nucleosides more water-soluble

than free bases

glycosidic bond

NucleotidesNucleoside phosphates

Functions of Nucleotides Nucleoside 5'-triphosphates are carriers of

energy Bases serve as recognition units Cyclic nucleotides are signal molecules and

regulators of cellular metabolism and reproduction

ATP is central to energy metabolism GTP drives protein synthesis CTP drives lipid synthesis UTP drives carbohydrate metabolism

Nucleic Acids - Polynucleotides

Polymers linked 3' to 5' by phosphodiester bridges

Ribonucleic acid and deoxyribonucleic acid

Sequence is always read 5' to 3' In terms of genetic information, this corre

sponds to "N to C" in proteins

Nucleotide monomers are joined by 3’-5’ phosphodiester linkages to form nucleic acid (polynucleotide) polymers

Classes of Nucleic Acids

DNA - one type, one purpose RNA - several types, several purposes

ribosomal RNA - the basis of structure and function of ribosomes

messenger RNA - carries the message transfer RNA - carries the amino acidsmicroRNA - regulates gene expression

Messenger RNA

Transcription product of DNA In prokaryotes, a single mRNA contains th

e information for synthesis of many proteins

In eukaryotes, a single mRNA codes for just one protein, but structure is composed of introns and exons

Eukaryotic mRNA DNA is transcribed to produce heterogeneous nuclear RNA

mixed introns and exons with poly A intron - intervening sequence

exon - coding sequence poly A tail - is important for the nuclear export, translation, and stability of mRNA.

Splicing produces final mRNA without introns

Ribosomal RNA

Ribosomes are about 2/3 RNA, 1/3 protein rRNA serves as a scaffold for ribosomal pr

oteins 23S rRNA in E. coli is the peptidyl transfer

ase

Transfer RNA Small polynucleotide chains - 73 to

94 residues each Several bases usually methylated Each a.a. has at least one unique t

RNA which carries the a.a. to the ribosome

3'-terminal sequence is always CCA-a.a.

Aminoacyl tRNA molecules are the substrates of protein synthesis

DNA & RNA Differences?Why does DNA contain thymine?

Cytosine spontaneously deaminates to form uracil

Repair enzymes recognize these "mutations" and replace these Us with Cs

But how would the repair enzymes distinguish natural U from mutant U?

Nature solves this dilemma by using thymine (5-methyl-U) in place of uracil

DNA & RNA Differences?

Why is DNA 2'-deoxy and RNA is not? Vicinal -OH groups (2' and 3') in RNA

make it more susceptible to hydrolysis DNA, lacking 2'-OH is more stable This makes sense - the genetic material

must be more stable RNA is designed to be used and then

broken down

The Structure of DNA

Diameter of 2 nm Length of 1.6 million nm (E. coli) Compact and folded (E. coli cell is only 2000 n

m long) Eukaryotic DNA wrapped around histone protei

ns to form nucleosomes Base pairs: A-T, G-C

DNA Structure level 1- Linear array of

nucleotides Structure level 2- double helix Structure level 3- Super-coiling,

stem-loop formation Structure level 4- Packaging into

chromatin

The DNA Double Helix

Stabilized by hydrogen bonds "Base pairs" arise from hydrogen bonds Erwin Chargaff had the pairing data, but di

dn't understand its implications Rosalind Franklin's X-ray fiber diffraction d

ata was crucial Francis Crick knew it was a helix James Watson figured out the H-bonds

Base pairing evident in DNA compositions

Bases from two adjacent DNA strands can hydrogen bond

•Guanine pairs with cytosine

•Adenine pairs with thymine

H-bonding of adjacent antiparallel DNA strands form double helix structure

Properties of DNA Double Helix

Hydrophillic sugar phosphate backbone winds around outside of helix

Noncovalent interactions between upper and lower surfaces of base-pairs (stacking) forms a closely packed hydrophobic interior.

Hydrophobic environment makes H-bonding between bases stronger (no competition with water)

Cause the sugar-phosphate backbone to twist.

View down the Double Helix

Sugar-phosphatebackbone

Hydrophobic Interior with base

pair stacking

Factors stabilizing DNA double Helix

Hydrophobic interactions – burying hydrophobic purine and pyrimidine rings in interior

Stacking interactions – van der Waals interactions between stacked bases.

Hydrogen Bonding – H-bonding between bases

Charge-Charge Interactions – Electrostatic repulsions of negatively charged phosphate groups are minimized by interaction with cations (e.g. Mg2+)

DNA Secondary structure

DNA is double stranded with antiparallel strands

Right hand double helix Three different helical forms (A, B an

d Z DNA.

Comparison of A, B, Z DNA• A: right-handed, short and broad, 2.3 A, 11 bp

per turn • B: right-handed, longer, thinner, 3.32 A, 10 bp

per turn • Z: left-handed, longest, thinnest, 3.8 A, 12 bp

per turn

A-DNA B-DNA Z-DNA

Z-DNA• Found in G:C-rich regions of DNA

• G goes to syn conformation

• C stays anti but whole C nucleoside (base and sugar) flips 180 degrees

DNA sequence Determines Melting Point

Double Strand DNA can be denatured by heat (get strand separation)

Can determine degree of denturation by measuring absorbance at 260 nm.

Conjugated double bonds in bases absorb light at 260 nm.

Base stacking causes less absorbance.

Increased single strandedness causes increase in absorbance

DNA sequence Determines Melting Point

Melting temperature related to G:C and A:T content.

3 H-bonds of G:C pair require higher temperatures to denture than 2 H-bonds of A:T pair.

DNA Structure Level 3

Super coiling Cruciform structures (cross shape)

Supercoils• In duplex DNA, ten bp per turn of helix (relaxed for

m)• DNA helix can be over-wound.• Over winding of DNA helix can be compensated by s

upercoiling.• Supercoiling prevalent in circular DNA molecules an

d within local regions of long linear DNA strands• Enzymes called topoisomerases or gyrases can intr

oduce or remove supercoils• In vivo most DNA is negatively supercoiled.• Therefore, it is easy to unwind short regions of the

molecule to allow access for enzymes

Each super coil compensates for one + or – turn of the double helix

•Cruciforms occur in palindromic regions of DNA

•Can form intrachain base pairing

•Negative supercoiling may promote cruciforms

DNA Structure level 4

In chromosomes, DNA is tightly associated with proteins

Chromosome Structure

• Human DNA’s total length is ~2 meters!• This must be packaged into a nucleus that

is about 5 micrometers in diameter• This represents a compression of more th

an 100,000!• It is made possible by wrapping the DNA a

round protein spools called nucleosomes and then packing these in helical filaments

Nucleosome Structure• Chromatin, the nucleoprotein comple

x, consists of histones and nonhistone chromosomal proteins

• major histone proteins: H1, H2A, H2B, H3, and H4

• Histone octamers are major part of the “protein spools”

• Nonhistone proteins are regulators of gene expression

Histones H2A, H2B, H3 and H4 are known as the core histones, while histones H1 are known as the linker histones.

•4 major histone (H2A, H2B, H3, H4) proteins for octomer

•200 base pair long DNA strand winds around the octomer

•146 base pair DNA “spacer separates individual nucleosomes

•H1 protein involved in higher-order chromatin structure.

•Without H1, Chromatin looks like beads on string

Solenoid Structure of Chromatin

Hydrolysis of Nucleic Acids

RNA is resistant to dilute acid DNA is depurinated by dilute acid DNA is not susceptible to base RNA is hydrolyzed by dilute base

Restriction Enzymes Bacteria have learned to "restrict" the possibility of attack fro

m foreign DNA by means of "restriction enzymes" Type II restriction enzymes cleave DNA chains at selected sit

es Type II restriction enzymes cut DNA about 20-30 base pairs

after the recognition site. Type I enzymes cut at a site that differs, and is a random dist

ance (at least 1000 bp) away, from their recognition site. Enzymes may recognize 4, 6 or more bases in selecting sites

for cleavage An enzyme that recognizes a 6-base sequence is a "six-cutte

r"

Type II Restriction Enzymes

No ATP requirement Recognition sites in dsDNA usually have a

2-fold axis of symmetry Cleavage can leave staggered or "sticky"

ends or can produce "blunt” ends

Type II Restriction Enzymes Names use 3-letter italicized

code: 1st letter - genus; 2nd,3rd - s

pecies Following letter denotes strain EcoRI is the first restriction en

zyme found in the R strain of E. coli

DNA sequencing---Chain Termination Method

• Based on DNA polymerase reaction • 4 separate rxns• Each reaction mixture contains dATP, dGTP, dCT

P and dTTP• Each reaction also contains a small amount of on

e dideoxynucleotide (ddATP, ddGTP, ddCTP and ddTTP).

• Each of the 4 dideoxynucleotides are labeled with a different fluorescent dye.

• Dideoxynucleotides missing 3’-OH group. Once incorporated into the DNA chain, chain elongation stops)

N

NN

N

NH2

O

H

HH

HH

NH

N

N

O

NH2N

O

H

HH

HHO

PO

O

HO

O-

N

NN

N

NH2

O

HO

HH

HH

PO

O

O-

NH

N

N

O

NH2N

O

H

HH

HHO

PO

O

HO

O-

NH

N

N

O

NH2N

O

H

HH

HHOH

OH

OH

PHO

O

O-

NH

N

N

O

NH2N

O

H

HH

HHOH

OH

PO

O

PO

O

O-

No Chain Elongation

Chain Termination Method

• Run each reaction mixture on electrophoresis gel • Short fragments go to bottom, long fragments on

top • Read the "sequence" from bottom of gel to top • Convert this "sequence" to the complementary

sequence

• Now read from the other end and you have the sequence you wanted - read 5' to 3'

AUTOMATED DNA SEQUENCING

The polymerase chain reaction (PCR) is a method to rapidly amplify sequences of DNA.

Lab for next week

Activity Determination of Serum Glutamate Pyruvate Transaminase