Download - Lecture 6
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Lecture 6Nucleotides and Nucleic Acids
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Clarification for the previous lessons 2,3-disphosphoglycerate (2,3-DPG ) = 2,
3-bisphosphoglycerate (2,3-BPG) Hemoglobin saturation curve =
oxygen–hemoglobin dissociation curve
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oxygen–hemoglobin dissociation curve
CO2 ?
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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)
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Other factors interfering with O2 loading: Carbon monoxide - displaces oxygen from hemoglobin Methemoglobinemia Fe2+ → Fe3+ (doesn't combine with O2)
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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?
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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.
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Nucleic Acids
Compound contained C, N, O, and high amount of P.
Was an acid compound found in nuclei therefore named nucleic acid
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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.
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Nitrogenous Bases
Pyrimidines Cytosine (DNA, RNA) Uracil (RNA) Thymine (DNA)
Purines Adenine (DNA, RNA) Guanine (DNA, RNA)
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Nitrogenous Bases
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Properties of Pyrimidines and Purines Keto-enol tautomerism
Strong absorbance of UV light
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absorbance of UV light
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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
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L-ribose and L-deoxyribose not found in nature
D-amino acids is rare.
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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
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glycosidic bond
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NucleotidesNucleoside phosphates
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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
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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
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Nucleotide monomers are joined by 3’-5’ phosphodiester linkages to form nucleic acid (polynucleotide) polymers
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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Base pairing evident in DNA compositions
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Bases from two adjacent DNA strands can hydrogen bond
•Guanine pairs with cytosine
•Adenine pairs with thymine
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H-bonding of adjacent antiparallel DNA strands form double helix structure
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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.
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View down the Double Helix
Sugar-phosphatebackbone
Hydrophobic Interior with base
pair stacking
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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+)
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DNA Secondary structure
DNA is double stranded with antiparallel strands
Right hand double helix Three different helical forms (A, B an
d Z DNA.
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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
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A-DNA B-DNA Z-DNA
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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
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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
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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.
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DNA Structure Level 3
Super coiling Cruciform structures (cross shape)
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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
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Each super coil compensates for one + or – turn of the double helix
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•Cruciforms occur in palindromic regions of DNA
•Can form intrachain base pairing
•Negative supercoiling may promote cruciforms
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DNA Structure level 4
In chromosomes, DNA is tightly associated with proteins
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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
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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
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Histones H2A, H2B, H3 and H4 are known as the core histones, while histones H1 are known as the linker histones.
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•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
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Solenoid Structure of Chromatin
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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
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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"
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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
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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
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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)
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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
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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'
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AUTOMATED DNA SEQUENCING
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The polymerase chain reaction (PCR) is a method to rapidly amplify sequences of DNA.
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Lab for next week
Activity Determination of Serum Glutamate Pyruvate Transaminase