7/29/2019 Chem 4311 Chapter10 12 Nucleic Acid
1/36
Chapter 10-12Nucleotides and Nucleic Acids
EXAM - October 19
Ins tructor: Dr. Khairu l I AnsariOffice: 316CPBPhone: 817-272-0616email: [email protected] hours 12 am 1:30 pm Tuesday &.Thursday
CHEM 4311General Biochemistry I
Fall 2012
Chapter 10
Nucleotides and Nucleic Acids
We have discovered thesecret of life.Francis Crick, to patrons ofThe Eagle, a pub inCambridge, England(1953)
Francis Crick (right) andJames Watson (left)point out features of their
model for the structure ofDNA.
Information Transfer in Cells
Figure 10.1 Thefundamental process ofinformation transfer incells.
10.1 What Are the Structure and Chemistry ofNitrogenous Bases?
Know the basic structures
Pyrimidines
Cytosine (DNA, RNA)Uracil (RNA)
Thymine (DNA)
Purines
Adenine (DNA, RNA)
Guanine (DNA, RNA)
10.1 What Are the Structure and Chemistry ofNitrogenous Bases?
Figure 10.2 (a) The pyrimidine ring system; by convention,atoms are numbered as indicated.
(b) The purine ring system; atoms numbered as shown.
7/29/2019 Chem 4311 Chapter10 12 Nucleic Acid
2/36
10.1 What Are the Structure and Chemistry ofNitrogenous Bases?
Figure 10.3 The common pyrimidine bases cytosine, uracil, and thymine in the tautomeric formspredominant at pH 7.
10.1 What Are the Structure and Chemistry ofNitrogenous Bases?
Figure 10.4 The common purine bases adenine andguanine in the tautomeric forms predominant at pH 7.
The Properties of Pyrimidines and Purines Can Be Traced to TheirElectron-Rich Nature
The aromaticity and electron-rich nature ofpyrimidines and purines enable them to undergoketo-enol tautomerism
The keto tautomers of uracil, thymine, and guaninepredominate at pH 7
By contrast, the enol form of cytosine predominatesat pH 7
Protonation states of the nitrogens determineswhether they can serve as H-bond donors oracceptors
Aromaticity also accounts for strong absorption of UVlight
The Properties of Pyrimidines and Purines Can Be Traced toTheir Electron-Rich Nature
Figure 10.6 The keto-enol tautomerism of uracil.
The Properties of Pyrimidines and Purines Can Be Traced toTheir Electron-Rich Nature
Figure 10.7 The tautomerization of the purine guanine.
The Properties of Pyrimidines and Purines Can Be Traced to TheirElectron-Rich Nature
Figure 10.8 The UV absorption spectra of the commonribonucleotides.
7/29/2019 Chem 4311 Chapter10 12 Nucleic Acid
3/36
The Properties of Pyrimidines and Purines Can Be Traced toTheir Electron-Rich Nature
Figure 10.8 The UV absorption spectra of the commonribonucleotides.
10.2 What Are Nucleosides?
Structures to Know
Nucleosides are compounds formed when a base islinked to a sugar via a glycosidic bond
The sugars are pentoses
D-ribose (in RNA)
2-deoxy-D-ribose (in DNA) The difference - 2'-OH vs 2'-H
This difference affects secondary structure andstability
10.2 What Are Nucleosides?
Figure 10.9 The linear and cyclic (furanose) forms ofribose.
10.2 What Are Nucleosides?
Figure 10.9 The linear and cyclic (furanose) forms ofdeoxyribose.
10.2 What Are Nucleosides?
The base is linked to the sugar via a glycosidicbond
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 apurine
Conformation can be syn or anti
Sugars make nucleosides more water-solublethan free bases
10.2 What Are Nucleosides?
Figure 10.10 The common ribonucleosides.
7/29/2019 Chem 4311 Chapter10 12 Nucleic Acid
4/36
Adenosine: A Nucleoside with Physiological Activity
Adenosine functions as anautacoid, or local hormone, and
neuromodulator. Circulating in the bloodstream, it
influences blood vessel dilation,smooth muscle contraction,neurotransmitter release, and fatmetabolism.
Adenosine is also a sleepregulator. Adenosine rises duringwakefulness, promoting eventualsleepiness.
Caffeine promotes wakefulnessby blocking binding of adenosineto its neuronal receptors.
10.3 What Is the Structure and Chemistry ofNucleotides?
Figure 10.11 Structures of the four common ribonucleotides AMP, GMP, CMP, and UMP. Also shown: 3 -AMP.
10.3 What Is the Structure and Chemistry ofNucleotides?
Figure 10.13 Formation of ADP and ATP by the succesiveaddition of phosphate groups via phosphoric anhydridelinkages. Note that the reaction is a dehydration synthesis.
10.3 What Is the Structure and Chemistry ofNucleotides?
Figure 10.13 Formation of ADP and ATP by the succesiveaddition of phosphate groups via phosphoric anhydridelinkages. Note that the reaction is a dehydration synthesis.
Nucleoside 5'-Triphosphates Are Carriers ofChemical Energy
Nucleoside 5'-triphosphates are indispensableagents in metabolism because their phosphoricanhydride bonds are a source of chemical energy
Bases serve as recognition units Cyclic nucleotides are signal molecules and
regulators of cellular metabolism andreproduction ATP is central to energy metabolism GTP drives protein synthesis CTP drives lipid synthesis UTP drives carbohydrate metabolism
Nucleoside 5'-Triphosphates Are Carriers ofChemical Energy
Figure 10.14 Phosphoryl, pyrophosphoryl, and nucleotidylgroup transfer, the major biochemical reactions ofnucleotides. Phosphoryl group transfer is shown here.
7/29/2019 Chem 4311 Chapter10 12 Nucleic Acid
5/36
Nucleoside 5'-Triphosphates Are Carriers ofChemical Energy
Figure 10.14 Phosphoryl, pyrophosphoryl, andnucleotidyl group transfer, the major biochemicalreactions of nucleotides. Pyrophosphoryl grouptransfer is shown here.
Nucleoside 5'-Triphosphates Are Carriers ofChemical Energy
Figure 10.14 Phosphoryl, pyrophosphoryl, andnucleotidyl group transfer, the major biochemicalreactions of nucleotides. Nucleotidyl group transfer isshown here.
10.4 What Are Nucleic Acids?
Nucleic acids are linear polymers of nucleotideslinked 3' to 5' by phosphodiester bridges
Ribonucleic acid and deoxyribonucleic acid Know the shorthand notations Sequence is always read 5' to 3' In terms of genetic information, this
corresponds to "N to C" in proteins
10.4 What Are Nucleic Acids?
Figure 10.15 3',5'-Phosphodiester bridges linknucleotides together to formpolynucleotide chains. The 5'-ends of the chains are at thetop; the 3'-ends are at thebottom. RNA is shown here.
10.4 What Are Nucleic Acids?
Figure 10.15 3 ,5 -phosphodiester bridges linknucleotides together to formpolynucleotide chains. The
5 -ends of the chains are atthe top; the 3 -ends are atthe bottom. DNA is shownhere.
10.5 What Are the Different Classes of NucleicAcids?
DNA - one type, one purpose
RNA - 3 (or 4) types, 3 (or 4) purposes
ribosomal RNA - the basis of structure and functionof ribosomes
messenger RNA - carries the message for protein
synthesistransfer RNA - carries the amino acids for protein
synthesis
Others: Small nuclear RNA
Small non-coding RNAs
7/29/2019 Chem 4311 Chapter10 12 Nucleic Acid
6/36
10.5 What Are the Different Classes of NucleicAcids?
Figure 10.16 Theantiparallel nature of theDNA double helix. The twochains have oppositeorientations.
The DNA Double Helix
The double helix is stabilized by hydrogen bonds
"Base pairs" arise from hydrogen bondsA-T; G-C
Erwin Chargaff had the pairing data, but didn'tunderstand its implications
Rosalind Franklin's X-ray fiber diffraction datawas crucial
Francis Crick showed that it was a helix James Watson figured out the H bonds
The Base Pairs Postulated by Watson
Figure 10.17 The Watson-Crick base pairs A:T and G:C.A:T is shown here.
The Base Pairs Postulated by Watson
Figure 10.17 The Watson-Crick base pairs A:T and G:C.
G:C is shown here.
The Structure of DNA
An antiparallel double helix
Diameter of 2 nm Length of 1.6 million nm (E. coli) Compact and folded (E. colicell is only 2000
nm long) Eukaryotic DNA wrapped around histone
proteins to form nucleosomes Base pairs: A-T, G-C
The Structure of DNA
Figure 10.18 Replication of DNAgives identical progeny moleculesbecause base pairing is themechanism that determines thenucleotide sequence of each newlysynthesized strand.
7/29/2019 Chem 4311 Chapter10 12 Nucleic Acid
7/36
Digestion of the E. colicell wall releases thebacterial chromosome
Figure 10.19 The chromosome is shown surrounding the cell.
Do the Properties of DNA Invite PracticalApplications?
The molecular recognition between DNA strandscan create a molecule with mechanical propertiesdifferent from single-stranded DNA
DNA double helices are relatively rigid rods DNA chains have been used to construct
nanomachines capable of simple movementssuch as rotation and pincerlike motions
More elaborate DNA-based devices can act asmotors walking along DNA tracks
The construction of DNA tweezers is describedon the following slide
Do the Properties of DNA Invite PracticalApplications?
DNA tweezers a simple
DNA nanomachine.
Messenger RNA Carries the Sequence Informationfor Synthesis of a Protein
Transcription product of DNA
In prokaryotes, a single mRNA contains theinformation for synthesis of many proteins
In eukaryotes, a single mRNA codes for justone protein, but structure is composed ofintrons and exons
Messenger RNA Carries the Sequence Information forSynthesis of a Protein
Figure 10.20 Transcription and translation of mRNAmolecules in prokaryotic versus eukaryotic cells.
In prokaryotes, a single mRNA molecule may contain theinformation for the synthesis of several polypeptidechains within its nucleotide sequence.
Messenger RNA Carries the Sequence Information forSynthesis of a Protein
Figure 10.20 Transcription and translation of mRNA moleculesin prokaryotic versus eukaryotic cells.Eukaryotic mRNAs encode only one polypeptide but are morecomplex.
7/29/2019 Chem 4311 Chapter10 12 Nucleic Acid
8/36
Eukaryotic mRNA
DNA is transcribed to produce heterogeneousnuclear RNA (hnRNA)
mixed introns and exons with poly A
intron = intervening sequence
exon = coding sequence
poly A tail - stability?
Splicing produces final mRNA without introns
Ribosomal RNA Provides the Structural andFunctional Foundation for Ribosomes
Ribosomes are about 2/3 RNA, 1/3 protein rRNA serves as a scaffold for ribosomal proteins The different species of rRNA are referred to
according to their sedimentation coefficients rRNAs typically contain certain modified nucleotides,
including pseudouridine and ribothymidylic acid The role of ribosomes in biosynthesis of proteins is
treated in detail in Chapter 30 Briefly: the genetic information in the nucleotide
sequence of mRNA is translated into the amino acidsequence of a polypeptide chain by ribosomes
Ribosomal RNA Provides the Structural andFunctional Foundation for Ribosomes
Figure 10.21 Ribosomal RNAhas a complex secondarystructure due to manyintrastrand H bonds. The grayline here traces apolynucleotide chain consistingof more than 1000 nucleotides.Aligned regions represent H-bonded complementary basesequences.
Ribosomal RNA Provides the Structural andFunctional Foundation for Ribosomes
Figure 10.22 The organization and composition of ribosomes.
Transfer RNAs Carry Amino Acids to Ribosomesfor Use in Protein Synthesis
Small polynucleotide chains - 73 to 94 residues each Several bases usually methylated Each a.a. has at least one unique tRNA which carries
the a.a. to the ribosome 3'-terminal sequence is always CCA-3-OH. The a.a.
is attached in ester linkage to this 3-OH. Aminoacyl tRNA molecules are the substrates of
protein synthesis
Transfer RNAs Carry Amino Acids to Ribosomes for Usein Protein Synthesis
Figure 10.24 Transfer RNA alsohas a complex secondarystructure due to many intrastrandhydrogen bonds. The black linesrepresent base-pairednucleotides in the sequence.
7/29/2019 Chem 4311 Chapter10 12 Nucleic Acid
9/36
The RNA World and Early Evolution
Thomas Cech and Sidney Altman showed that RNAmolecules are not only informational they can also becatalytic
This gave evidence to the postulate by Francis Crickand others that prebiotic evolution depended on self-replicating, catalytic RNAs
But what was the origin of the nucleotides? A likely source may have been conversion of
aminoimidazolecarbonitrile to adenine And glycolaldehyde could combine with other
molecules to form ribose Adenine and glycolaldehyde exist in outer space
The RNA World and Early Evolution
Aminoimidazolecarbonitrile is a pentamer of HCN andmay be a celestial precursor of adenine.
The RNA World and Early Evolution
Glycolaldehyde has been detected at thecenter of the Milky Way and could be aprecursor of ribose and glucose.
The Chemical Differences Between DNA and RNAHave Biological Significance
Two fundamental chemical differencesdistinguish DNA from RNA:
DNA contains 2-deoxyribose instead of ribose
DNA contains thymine instead of uracil
The Chemical Differences Between DNA and RNAHave Biological Significance
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 moresusceptible 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
7/29/2019 Chem 4311 Chapter10 12 Nucleic Acid
10/36
10.6 Are Nucleic Acids Susceptible to Hydrolysis?
RNA is resistant to dilute acid
DNA is depurinated by dilute acid DNA is not susceptible to base
RNA is hydrolyzed by dilute base
See Figure 10.29 for mechanism
10.6 Are Nucleic Acids Susceptible to Hydrolysis?
Figure 10.27 Alkaline hydrolysis of RNA. Nucleophilicattach by OH- on the P atom leads to 5'-phosphoestercleavage.
10.6 Are Nucleic Acids Susceptible to Hydrolysis?
Figure 10.27 Alkaline hydrolysis of RNA. Nucleophilic attack byOH- on the P atom leads to 5'-phosphoester cleavage. Random
hydrolysis of the cyclic phosphodiester intermediate gives amixture of 2'- and 3'-nucleoside monophosphate products.
10.6 Are Nucleic Acids Susceptible to Hydrolysis?
Figure 10.27 Alkaline hydrolysis of RNA. Random hydrolysis ofthe cyclic phosphodiester intermediate gives a mixture of 2'- and
3'-nucleoside monophosphate products.
10.6 Are Nucleic Acids Susceptible to Hydrolysis?
Figure 10.28 Cleavage in polynucleotide chains. Cleavageon the a side leaves the phosphate attached to the 5'-position of the adjacent nucleotide. b-side hydrolysis yields3'-phosphate products.
10.6 Are Nucleic Acids Susceptible to Hydrolysis?
Figure 10.28 Cleavage in polynucleotide chains.Cleavage on the a side leaves the phosphate attached tothe 5'-position of the adjacent nucleotide.
7/29/2019 Chem 4311 Chapter10 12 Nucleic Acid
11/36
10.6 Are Nucleic Acids Susceptible to Hydrolysis?
Figure 10.28 Cleavage in polynucleotide chains.b-side hydrolysis yields 3'-phosphate products, amongothers.
Restriction Enzymes
Bacteria have learned to "restrict" the
possibility of attack from foreign DNA bymeans of "restriction enzymes"
Type II and III restriction enzymes cleave DNAchains at selected sites
Enzymes may recognize 4, 6 or more bases inselecting sites for cleavage
An enzyme that recognizes a 6-base sequenceis a "six-cutter"
Type II Restriction Enzymes
No ATP requirement
Recognition sites in dsDNA have a 2-fold axis ofsymmetry
Cleavage can leave staggered or "sticky" ends orcan produce "blunt ends
Type II Restriction Enzymes
Names use 3-letter italicized code:
1st letter - genus; 2nd,3rd - species
Following letter denotes strain
EcoRI is the first restriction enzyme isolated fromthe R strain ofE. coli
Cleavage Sequences of Restriction Endonucleases Cleavage Sequences of Restrict ion Endonucleases
7/29/2019 Chem 4311 Chapter10 12 Nucleic Acid
12/36
Restriction Mapping of DNA
Figure 10.29 Restriction mapping analysis.
Chapter 11
Structure of Nucleic Acids
Chapter 11
The Structure of DNA:A melody for the eye of
the intellect, with not a notewasted.Horace Freeland JudsonThe Eighth Day of Creation
11.1 How Do Scientists Determine the PrimaryStructure of Nucleic Acids?
Two simple tools have made nucleic acidsequencing easier than polypeptidesequencing:
The type II restriction endonucleases that cleaveDNA at specific oligonucleotide sites
Gel electrophoresis, which is capable of separatingnucleic acid fragments that differ from oneanother in length by just a single nucleotide
11.1 How Do Scientists Determine the PrimaryStructure of Nucleic Acids?
Chain termination method (dideoxy method),developed by Frederick Sanger is the basis for mostDNA sequencing currently.
The method takes advantage of the DNA polymerasereaction, which copies a DNA strand in
complementary fashion to form a new second strand
11.1 How Do Scientists Determine the PrimaryStructure of Nucleic Acids?
Figure 11.1 DNA replication yields twodaughter DNA duplexes identical to theparental DNA molecule.
7/29/2019 Chem 4311 Chapter10 12 Nucleic Acid
13/36
11.1 How Do Scientists Determine the PrimaryStructure of Nucleic Acids?
Figure 11.2 Primed synthesis of a DNA template byDNA polymerase, using the four deoxynucleosidetriphosphates as the substrates.
11.1 How Do Scientists Determine the PrimaryStructure of Nucleic Acids?
DNA is a double-helical molecule Each strand of the helix must be copied incomplementary fashion by DNA polymerase
Each strand is a template for copying DNA polymerase requires template and primer Primer: an oligonucleotide that pairs with the
end of the template molecule to form dsDNA DNA polymerases add nucleotides in 5'-3'
direction
Chain Termination Method
Primer extension: A template DNA base-paired witha complementary primer is copied by DNApolymerase in the presence of dATP, dCTP, dGTP,dTTP
Solution contains small amounts of the fourdideoxynucleotide analogs of these substrates, eachof which contains a distinctive fluorescent tag,illustrated here as:
Orange for ddATP
Blue for ddCTP
Green for ddGTP
Red for ddTTP
Occasional incorporation of a dideoxynucleotideterminates further synthesis of that strand
Figure 11.3 The chain
termination method ofDNA sequencing.
Chain Termination Method
Most of the time, the polymerase uses normalnucleotides and DNA molecules grow normally
Occasionally, the polymerase uses adideoxynucleotide, which prevents furtherextension when added to the growing chain
Random insertion of dd-nucleotides leaves(optimally) at least a few chains terminated atevery occurrence of a given nucleotide
Chain Termination Method
Reaction mixtures can be separated by capillaryelectrophoresis
Short fragments go to bottom, long fragments ontop
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'
7/29/2019 Chem 4311 Chapter10 12 Nucleic Acid
14/36
The set of terminated strands can be separatedby capillary electrophoresis
High-Throughput DNA Sequencing by the Light ofFireflies
The importance of DNA sequence information has
motivated development of more rapid and efficientDNA sequencing technologies
454 Technology relies on DNA polymerase but doesnot involve chain termination
Multiple copies of template DNA molecules areimmobilized on microscopic beads
Reagents for primed synthesis are passed over thebeads
Pyrophosphate release is monitored by lightemission via ATP sulfurylase and luciferase reactions
High-Throughput DNA Sequencing by the Light ofFireflies
DNA polymerase action produces PPi:(NMP)n + NTP (NMP)n+1 + PPi
ATP sulfurylase: PPi + APS ATP + SO42-
Luciferase:ATP + luciferin + O2 AMP + PPi + CO2 + oxyluciferin + light
Structures of luciferin and oxyluciferin. Light detectionconfirms that addition of a dNMP by primed synthesis hasoccurred.
High-Throughput DNA Sequencing by the Lightof Fireflies
Figure 11.4
Emerging Technologies to Sequence DNA are Based onSingle-Molecule Sequencing Strategies
Growing demand for sequence information is driving thedevelopment of faster and cheaper methods of DNAsequencing
Most promising are the single-molecule strategies that donot rely on Sanger-based primed synthesis of strandscomplementary to prepared DNA samples
One technique involves passing a single strand of DNAthrough a graphene monolayer pore, measuring thechange in electrical conductance (ion flow) through thepore
Each base alters electrical conductance in a subtle butdifferent way, facilitating the reading of sequence
Figure 11.5 DNA Sequencing through a pore in agraphene monolayer
7/29/2019 Chem 4311 Chapter10 12 Nucleic Acid
15/36
11.2 What Sorts of Secondary Structures Can Double-Stranded DNA Molecules Adopt?
Polynucleotide strands are flexible
Each deoxyribose-phosphate segment of thebackbone has six degrees of freedom (Fig 11.4a)
Furanose rings are not planar but instead adoptpuckered conformations, four of which are shown inFigure 11.4b
A seventh degree of freedom per nucleotide arisesbecause of free rotation about the C1'-N glycosidicbond
This freedom allows the plane of the base to rotaterelative to the path of the polynucleotide strand
11.2 What Sorts of Secondary Structures Can Double-Stranded DNA Molecules Adopt?
Figure 11.6 The six degrees of freedom in thedeoxyribose-PO4 units of the polynucleotide chain.
11.2 What Sorts of Secondary Structures Can Double-Stranded DNA Molecules Adopt?
Figure 11.6 Four puckered conformations of the furanoserings.
11.2 What Sorts of Secondary Structures CanDouble-Stranded DNA Molecules Adopt?
Figure 11.6 Free rotation about the C1'-N glycosidic bond.
Figure 11.7
(a) Double-stranded DNA as an imaginaryladderlike structure.(b) A simple right-handed twist converts theladder to a helix.
11.2 What Sorts of Secondary Structures Can Double-Stranded DNA Molecules Adopt?
The stability of the DNA double helix is due to:
Hydrogen bonds between base pairs
Electrostatic interactions mutual repulsion ofphosphate groups, which makes them most stable onthe helix exterior
Base-pair stacking interactions
Right-twist closes the gaps between base pairs to 3.4 A(0.34 nm) in B-DNA
See Figure 11.8 for details of DNA secondary structure
7/29/2019 Chem 4311 Chapter10 12 Nucleic Acid
16/36
The canonical base pairs
See Figure 11.8 The canonical A:T and G:C base pairs have nearly
identical overall dimensions
A and T share two H bonds
G and C share three H bonds
G:C-rich regions of DNA are more stable
Polar atoms in the sugar-phosphate backbonealso form H bonds
The canonical base pairs
Figure 11.8 Watson-
Crick A:T and G:C basepairs. All H-bonds inboth base pairs arestraight.
The canonical base pairs
Figure 11.8 Watson-Crick A:T and G:Cbase pairs. All H-bonds in both basepairs are straight.
Major and minor grooves
See Figures 11.8, 11.9
The "tops" of the bases (as we draw them) line the"floor" of the major groove
The major groove is large enough to accommodate analpha helix from a protein
Regulatory proteins (transcription factors) canrecognize the pattern of bases and the H-bondingpossibilities in the major groove
Major and minor grooves
Figure 11.9 Themajor and minorgrooves of B-DNA.
Double Helical Structures Can Adopt a Numberof Stable Conformations
The DNA double helix can adopt several stableconformations
Helical twist is the rotation of one base pair relativeto the next, around the axis of the double helix
Successive base pairs in B-DNA show a mean rotation
of 36 with respect to each other
Propellor twist involves rotation around a differentaxis, namely an axis perpendicular to the helix axis
See Figure 11.10
7/29/2019 Chem 4311 Chapter10 12 Nucleic Acid
17/36
Double Helical Structures Can Adopt a Numberof Stable Conformations
Figure 11.10 Helical twist and propellor twist in DNA(a) Helical twist: Successive base pairs in B-DNA show arotation with respect to each other.
Double Helical Structures Can Adopt a Numberof Stable Conformations
Figure 11.10 Helical twist and propellor twist in DNA.(b) Propellor twist: Rotation in this dimension allows thehydrophobic surfaces of bases to overlap better
Double Helical Structures Can Adopt a Number ofStable Conformations
Helical twist and propellor twist in DNA. (c) Each of thebases in a base pair shows positive propellor twist as viewed
along the N-glycosidic bond. Note how the hydrogen bondsbetween bases are distorted by this motion, yet remain intact.
Double Helical Structures Can Adopt a Number ofStable Conformations
Figure 11.11 The B-form of theDNA double helix. In B-form, thepitch (the distance required tocomplete one helical turn) is 3.4nm. Twelve base pairs of DNA areshown.
Double Helical Structures Can Adopt a Number ofStable Conformations
Figure 11.11 The B-form of theDNA double helix. In B-form, thepitch (the distance required tocomplete one helical turn) is 3.4
nm. Twelve base pairs of DNA areshown.
Double Helical Structures Can Adopt a Number ofStable Conformations
Figure 11.11 The A-form ofthe DNA double helix. Thepitch of the A-form helix is2.46; thus the A-form is ashorter, wider structure thanthe B-form. One turn in A-form DNA requires 11 bp tocomplete. Twelve base pairsare shown here.
7/29/2019 Chem 4311 Chapter10 12 Nucleic Acid
18/36
Double Helical Structures Can Adopt a Number ofStable Conformations
Figure 11.11 The A-form ofthe DNA double helix. Thepitch of the A-form helix is2.46; thus the A-form is ashorter, wider structure thanthe B-form. One turn in A-form DNA requires 11 bp tocomplete. Twelve base pairsare shown here.
Z-DNA
Discovered by Alex Rich
Found in G:C-rich regions of DNA G goes to syn conformation
C stays anti but whole C nucleoside (base andsugar) flips 180 degrees
Result is that G:C H bonds can be preserved in thetransition from B-form to Z-form!
Z-DNA is a Conformational Variation in theForm of a Left-Handed Double Helix
Figure 11.11 The Z-form of doublehelical DNA.
The N-glycosyl bonds of Gresidues in this alternatingcopolymer are rotated 180 withrespect to their conformation in B-DNA, so now the G nucleoside is inthe syn rather than the anticonformation.
The C residues remain in the antiform.
Because the G ring is flipped, the Cring must also flip to maintainnormal Watson-Crick base pairing.
Z-DNA is a Conformational Variation in the Formof a Left-Handed Double Helix
Figure 11.11 The Z-form ofdouble helical DNA.
The N-glycosyl bonds of Gresidues in this alternatingcopolymer are rotated 180with respect to theirconformation in B-DNA, sonow the purine ring is in thesyn rather than the anticonformation.
The C residues remain in theanti form.
Because the G ring is flipped,the C ring must also flip tomaintain normal Watson-Crickbase pairing.
Comparison of A, B, Z DNA
See Table 11.1
A: right-handed, short and broad, 2.3 , 11 bp perturn
B: right-handed, longer, thinner, 3.32 , 10 bp perturn
Z: left-handed, longest, thinnest, 3.8 , 12 bp perturn
See Figure 11.11
Comparison of A, B, Z DNA
7/29/2019 Chem 4311 Chapter10 12 Nucleic Acid
19/36
Comparison of B and Z DNA
Figure 11.12 Comparison of the deoxyguanosine conformationin B- and Z-DNA. It is topologically possible for G to go syn andthe C nucleoside to undergo rotation by 180 without breakingand re-forming the G:C hydrogen bonds.
The Change in Topological Relationships ofBase Pairs from B- to Z-DNA
Figure 11.13
DNA Methylation and Epigenetics
Methylation of cytosine residues (forming 5-methylcytosine) is essential for normal embryonicdevelopment
Cytosine methylation switches genes off, so that theinformation they encode is not expressed
Epigenetics is the study of heritable changes in thegenome that occur without a change in nucleotidesequence (such as cytosine methylation)
Epigenetic changes can influence expression of theinformation encoded by the genome
Intercalating Agents Distort the Double Helix
The double helix is a very dynamic structure
Because it is flexible, aromatic macrocycles flat hydrophobic molecules composed offused, heterocyclic rings, can slip between thestacked pairs of bases
The bases are force apart to accommodatethese intercalating agentsEthidium bromide
Acridine orange
Actinomycin D
Intercalating Agents Distort the Double Helix
Figure 11.14 Thestructures of ethidiumbromide, acridine orange,and actinomycin D, three
intercalating agents, andtheir effects on DNAstructure.
Alternative H-Bonding Interactions Give Riseto Novel DNA Structures
Cruciform structures arise frominverted repeats. In such structures,the normal interstrand base pairing isreplaced by intrastrand pairing.
Figure 11.15 Self-complementary invertedrepeats can rearrange to form H-bondedcruciform stem-loop structures. Cruciformsare not as stable as normal DNA, becausean unpaired segment must exist in the loop.
7/29/2019 Chem 4311 Chapter10 12 Nucleic Acid
20/36
Hoogsteen Base Pairs and DNA Multiplexes
Karst Hoogsteen found A:T and G:C base pairs that are different
from the canonical structures. In both A:T and G:C Hoogsteenbase pairs, the purine N-7 atom is an H-bond acceptor.
Figure 11.16 Hoogsteen base pairs: A:T (left) and C+:G (right).
Hoogsteen Base Pairs and DNA Multiplexes
Figure 11.17 Base triplets can form when a purine interactswith one pyrimidine by Hoogsteen base pairing and anotherby Watson-Crick base pairing.
H-DNA is Triplex DNA Made of One Purine-Rich Strandand Two Pyrimidine-Rich Strands
Figure 11.18 H-DNA.Pyrimidine-rich strandsare blue; purine-richstrands are green.
DNA Quadruplex Structures
Figure 11.19 G-quadruplexshowing the cyclic array ofguanines linked byHoogsteen hydrogen bonds.
G-quadruplexes are cyclicarrays of four G residuesunited through Hoogsteenbase pairing.
DNA Quadruplex Structures
Figure 11.19 Four G-richpolynucleotide strands inparallel alignment with all
bases in anti conformation.
DNA Quadruplex Structures
Figure 11.19 Antiparalleldimeric hairpinquadruplex formed byd(G4T4G4)2
7/29/2019 Chem 4311 Chapter10 12 Nucleic Acid
21/36
DNA Quadruplex Structures
Figure 11.19 Structure ofd(G4T4G4)2K
+ solved by X-ray crystallography. Twod(G4T4G4)2 strands cometogether as hairpins to forma G-quadruplex. Thebackbones of the twostrands are traced in violet.
11.3 Can the Secondary Structure of DNA BeDenatured and Renatured?
See Figure 11.20 When DNA is heated to 80 C or more, its UV
absorbance increases by 30-40% This hyperchromic shift reflects the unwinding of
the DNA double helix Stacked base pairs in native DNA absorb less light
due to , electron interactions When T is lowered, the absorbance drops, reflecting
re-establishment of the double helix and base-pairstacking
11.3 Can the Secondary Structure of DNA BeDenatured and Renatured?
Figure 11.18 Heat denaturation of DNA from various sources.
The Buoyant Density of DNA
Density gradient ultracentrifugation isa useful way to separate and purifynucleic acids.
The net movement of solute particles in an ultracentrifuge isthe result of two processes: diffusion (from regions of higher
concentration to regions of lower concentration) andsedimentation due to centrifugal force.
Single-Stranded DNA Can Renature to FormDNA Duplexes
Denatured DNA will renature to re-form theduplex structure if the denaturing conditionsare removed
Renaturation requires reassociation of the
DNA strands into a double helix, a processtermed reannealing
For this to occur, the strands must realign sothat their complementary bases are onceagain in register and the helix can be
zippered up
Single-Stranded DNA Can Renature to FormDNA Duplexes
Figure 11.21 Steps in thethermal denaturation andrenaturation of DNA.
7/29/2019 Chem 4311 Chapter10 12 Nucleic Acid
22/36
If DNA from two different species are mixed,denatured, and allowed to cool slowly, hybridduplexes may form, provided the DNA from onespecies is similar in sequence to the other
The degree of hybridization is a measure of thesequence similarity between the two species
25% of the DNA from a human forms hybrids withmouse DNA, implying some sequence similarity
Hybridization is a common procedure in molecularbiology for identifying specific genes and forrevealing evolutionary relationships
Nucleic Acid Hybridization: Different DNA Strands ofSimilar Sequence Can Form Hybrid Duplexes
Nucleic Acid Hybridization: Different DNA Strands ofSimilar Sequence Can Form Hybrid Duplexes
Figure 11.22 Solutions ofhuman DNA (red) and mouseDNA (blue) are mixed anddenatured; then, the singlestrands are allowed toreanneal.
About 25% of human DNAforms hybrid duplexes withmouse DNA.
11.4 Can DNA Adopt Structures of HigherComplexity?
In duplex DNA, there are ten bp per turn of helix
Circular DNA sometimes has more or less than 10 bpper turn - a supercoiled state
Enzymes called topoisomerases or gyrases canintroduce or remove supercoils
Cruciforms occur in palindromic regions of DNA
Negative supercoiling may promote cruciforms
Supercoils Are One Kind of Structural Complexityin DNA
Double-stranded circular DNA forms supercoils, if thestrands are underwound, or overwound.
Figure 11.23 Toroidal and interwound varieties of supercoiling.
Supercoiled DNA is characterized by a Linking Number(L), Twist (T), and Writhe (W)
Figure 11.24 Linking number (L) is sum of twist (T) and writhe (W)
Supercoiled DNA is characterized by a Linking Number(L), Twist (T), and Writhe (W)
Figure 11.24 Linking number (L) is sum of twist (T) and writhe (W)
7/29/2019 Chem 4311 Chapter10 12 Nucleic Acid
23/36
DNA Gyrase is a topoisomerase that introducesnegative supercoils into DNA
Figure 11.25 A model for the action ofbacterial DNA gyrase (topoisomeraseII).
Negative supercoils cause a torsionalstress on the molecule, so themolecule tends to unwind. Negativesupercoiling makes it easier toseparate DNA strands and access theinformation encoded by the sequence.
DNA Gyrase is a topoisomerase that introducesnegative supercoils into DNA
Figure 11.25 Conformationalchanges in the enzyme allow anintact region of the DNA duplexto pass between the cut ends.The cut ends are religated (3),and the covalently completeDNA duplex is released from theenzyme. The circular DNA nowcontains two negativesupercoils (4).
Negative Supercoiling has the Potential to CauseLocalized Unwinding in DNA
Figure 11.26 A 400-bp circular DNA molecule in differenttopological states: (a) relaxed, (b) negative supercoilsdistributed over the entire length, and (c) negative
supercoils creating a localized single-stranded region.
Negatively Supercoiled DNA Can Arrange into aToroidal State
Figure 11.28 The toroidal state is stabilized by wrappingaround proteins that serve as spools for the DNA ribbon .
11.5 What Is the Structure of EukaryoticChromosomes?
Human DNA s total length is ~2 meters!
This must be packaged into a nucleus that is about 5micrometers in diameter
This represents a compression of more than100,000!
It is made possible by wrapping the DNA aroundprotein spools called nucleosomes and thenpacking these in helical filaments
These filaments are thought to arrange in loopsassociated with the nuclear matrix
Nucleosomes Are the Fundamental StructuralUnit in Chromatin
Histones and nonhistone chromosomal proteins are the twoclasses of chromatin proteins. Five distinct histones areknown: H1, H2A, H2B, H3, and H4.
Pairs of histones H2A, H2B, H3, and H4 aggregate toform octameric core structures; the DNA helix is woundaround these core octamers, creating nucleosomes.
7/29/2019 Chem 4311 Chapter10 12 Nucleic Acid
24/36
Nucleosome Structure
Chromatin, the nucleoprotein complex,consists of histones and nonhistonechromosomal proteins
Histone octamer structure has been solved(without DNA by Moudrianakis, and with DNAby Richmond)
Nonhistone proteins are regulators of geneexpression
The Structure of the Nucleosome a HistoneOctamer wrapped with DNA
Figure 11.28
Structural Organization of Chromatin Gives Rise toChromosomes
The beads-on-a-string motif is the primarystructure of chromatin.
The secondary level of chromatin structure is the30-nm fiber, formed when an array of nucleosomesin a zig-zag pattern adopts a two-start helicalconformation (Figures 11.29a, b, c).
Higher levels of chromatin structural organization areachieved when the 30-nm fiber forms long loops of60-150,000 bp.
Electron microscopic analysis of human chromosome4 suggests that 18 such loops are then arranged
radially about the circumference of a single turn toform a miniband unit of the chromosome.
Higher Order Chromatin Organization
Figure 11.29a
Higher Order Chromatin Organization
Figure 11.29b
Higher Order Chromatin Organization
Figure 11.29c
7/29/2019 Chem 4311 Chapter10 12 Nucleic Acid
25/36
Higher-Order Structural Organization ofChromatin Gives Rise to Chromosomes
Figure 11.30 A model for chromosome structure, humanchromsome 4, showing nucleosomes in the beads on astring motif.
Higher-Order Structural Organization ofChromatin Gives Rise to Chromosomes
Figure 11.30 A model for chromosome structure, humanchromsome 4. The 30-nm fiber is created when the arrayof nucleosomes adopts a two-start helical conformation.
Higher-Order Structural Organization ofChromatin Gives Rise to Chromosomes
Figure 11.30 A model for chromosome structure, humanchromsome 4. The 30 nm filament forms long DNA loops ofvariable length, each containing on average between60,000 and 150,000 bp.
Higher-Order Structural Organization ofChromatin Gives Rise to Chromosomes
Figure 11.30 A model for chromosome structure, humanchromsome 4. Electron microscopic analysis ofchromosome 4 suggests that 18 loops are arranged
radially about the circumference of a single turn to form aminiband unit of the chromosome.
Higher-Order Structural Organization ofChromatin Gives Rise to Chromosomes
Figure 11.30 A model for chromosome structure, humanchromsome 4. Approximately a million minibands arearranged along a central axis in each of the chromatids ofchromosome 4 that form at mitosis.
SMC Proteins Aid Chromosome Organization andMediate Chromosome Dynamics
Figure 11.31 SMC protein architecture and func tion. SMCproteins range from 115 to 165 kD in size.
7/29/2019 Chem 4311 Chapter10 12 Nucleic Acid
26/36
SMC Proteins Aid Chromosome Organization andMediate Chromosome Dynamics
Figure 11.31 SMC protein architecture and function. Shownhere is the condensation of DNA into a coiled arrangementthrough SMC2/SMC4-mediated interactions.
11.6 Can Nucleic Acids Be SynthesizedChemically?
Laboratory synthesis of nucleic acids requires
orthogonal strategies. Functional groups on the monomeric units are
reactive and must be blocked.
Correct phosphodiester linkages must be made
Recovery at each step must high!
Solid-phase methods are used to satisfy some ofthese constraints.
Solid Phase Oligonucleotide Synthesis
The four-step cycle starts with the first base innucleoside form attached by its 3'-OH.
Then a dimethoxytrityl group blocks the 5'-OH ofthe first nucleoside while it is linked to a solidsupport by the 3'-OH.
Step 1: Detritylation by trichloroacetic acid exposesthe 5'-OH.
Step 2: In coupling reaction, second base is addedas a nucleoside phosphoramidate.
Solid Phase Oligonucleotide Synthesis
Step 3: Capping with acetic anhydride blocksunreacted 5'-OHs of N-1 from further reaction
Step 4: The phosphite linkage between N-1 andN-2 is reactive and is oxidized by aqueous iodineto form the desired, and more stable, phosphategroup
After the desired oligonucleotide has beenformed, it is freed of blocking groups, hydrolyzedfrom the resin, and purified by gelelectrophoresis
The four-step cycle is shown in Figure 11.32
Solid Phase Oligonucleotide Synthesis
The structure of the dimethoxytrityl group. (Atriphenylmethyl group is a trityl group.)
Figure 11.32
Solid Phase Oligonucleotide Synthesis
The DMTr protecting group can be removed bytrichloroacetic acid treatment.
Figure 11.32
7/29/2019 Chem 4311 Chapter10 12 Nucleic Acid
27/36
Solid Phase Oligonucleotide Synthesis
Figure 11.32 Benzoyl chloride can be used to protectNH2 functions
Solid Phase Oligonucleotide Synthesis
Figure 11.29
Figure 11.32 Isobutyryl chloride can also be used to protect-NH2 functions
Genes Can Be Synthesized Chemically11.7 What Are the Secondary and Tertiary
Structures of RNA?
The double-stranded structure of DNA imposes greatconstraints on its conformational possibilities
RNA molecules are typically single-stranded and thushave six to seven degrees of freedom per nucleotideunit
Thus RNA molecules have a much greater number ofconformational possibilities
Complementary sequences in RNA can join viaintrastrand base pairing
When the base pairing is not complete, a variety ofbulges and loops can form, including hairpin stem-loop structures
11.7 What Are the Secondary and TertiaryStructures of RNA?
Figure 11.33Bulges andloops formed inRNA when
alignedsequences arenot fullycomplementary
11.7 What Are the Secondary and TertiaryStructures of RNA?
A number of defined structural motifs recur withinthe loops of stem-loop structures, such as U-turns,tetraloops, and bulges
Regions where several stem-loop structures meet aretermedjunctions
Stems, loops, bulges, and junctions are the four basicsecondary structural elements in RNA
Other tertiary structural motifs arise from coaxialstacking, pseudoknot formation, and ribose zippers
7/29/2019 Chem 4311 Chapter10 12 Nucleic Acid
28/36
11.7 What Are the Secondary and TertiaryStructures of RNA?
Figure 11.34 Junctions and coaxial stacking in RNA.
11.7 What Are the Secondary and TertiaryStructures of RNA?
Figure 11.35 RNApseudoknots are formedwhen a single-strandedregion of RNA base-pairswith a hairpin loop.
Transfer RNA Adopts Higher-Order StructureThrough Intrastrand Base Pairing
In tRNA, with 73-94 nucleotides in a single chain, amajority of the bases are hydrogen- bonded to oneanother
Hairpin turns bring complementary stretches ofbases into contact
Extensive H-bonding creates four double helicaldomains, three capped by loops, one by a stem
Only one tRNA structure (alone) is known
Phenylalanine tRNA is "L-shaped"
Many non-canonical base pairs found in tRNA
Transfer RNA Adopts Higher-Order Structure ThroughIntrastrand Base Pairing
Figure 11.36 Ageneral diagram forthe structure of tRNA.
Transfer RNA Adopts Higher-Order StructureThrough Intrastrand Base Pairing
Figure 11.37 Tertiary structure intRNA arises from base-pairinginteractions between bases in theD loop with bases in the variableand TC loops, as shown here for
yeast phenylalanine tRNA.
Solid lines connect bases that arehydrogen-bonded when thiscloverleaf pattern is folded into thecharacteristic tRNA tertiarystructure (see Figure 11.38).
tRNA Tertiary Structure Arises From InterloopBase Pairing
Figure 11.38
7/29/2019 Chem 4311 Chapter10 12 Nucleic Acid
29/36
tRNA Tertiary Structure Arises From InterloopBase Pairing
Figure 11.38 Thethree-dimensionalstructure of yeastphenylalanine tRNA.The anticodon loop isat the bottom and theacceptor end is at thetop right.
Ribosomal RNA
Ribosomes synthesize proteins All ribosomes contain large and small subunits
rRNA molecules make up about 2/3 of ribosome
High intrastrand sequence complementarity leadsto extensive base-pairing
Secondary structure features seem to beconserved, whereas sequence is not
There must be common designs and functionsthat must be conserved
Ribosomal RNA also Adopts Higher-OrderStructure Through Intrastrand Base Pairing
These secondary structures of several 16S rRNAs are based on computeralignment of rRNAnucleotide sequences into optimal H-bonding segments.
Figure 11.39 Comparison of secondary structures of 16S-likerRNAs from several organisms.
rRNA Tertiary Structure
Figure 11.40 Detailedstructures of ribosomes havebeen revealed by X-raycrystallography andcryoelectron microscopy.
These images reveal details ofboth tertiary and quaternaryinteractions that occur whenribosomal proteins combinewith rRNAs to form thecomplete ribosome.
Riboswitches Act as Regulators of GeneExpression
Riboswitches, naturallyoccurring aptamers, areconserved regions of mRNAsthat reversibly bind specificmetabolites and coenzymes
and act as gene expressionregulators.
Figure 11.41
Chapter 12Recombinant DNA: Cloning and
Creation of Chimeric Genes
7/29/2019 Chem 4311 Chapter10 12 Nucleic Acid
30/36
12.1 What Does It Mean To Clone ?
Clone: a collection of molecules or cells, all
identical to an original molecule or cell
To "clone a gene" is to make many copies of it -for example, in a population of bacteria
Gene can be an exact copy of a natural gene
Gene can be an altered version of a natural gene
Recombinant DNA technology makes it possible
Plasmids Are Very Useful in Cloning Genes
Plamids are naturally-occurring
extrachromosomal DNA Plasmids are circular dsDNA
Plasmids can be cleaved by restriction enzymes,leaving sticky ends
Artificial plasmids can be constructed by linkingnew DNA fragments to the sticky ends of plasmid
These recombinant molecules can beautonomously replicated, and hence propagated
Cloning Vectors
Cloning vectors are plasmids that can be modified to
carry new genes
Plasmids useful as cloning vectors must have
a replicator (origin of replication)
a selectable marker (antibiotic resistance gene)
a cloning site (site where insertion of foreignDNA will not disrupt replication or inactivateessential markers)
Plasmids as Cloning Vectors
Figure 12.1 One ofthe first widely usedcloning vectors wasthe plasmid pBR322.
Note the antibioticresistance genes(ampr and tetr).
Virtually Any DNA Sequence Can Be Cloned
Nuclease cleavage at a restriction sitelinearizes the circular plasmid so that aforeign DNA fragment can be inserted.
Recombinant plasmids are hybridDNA molecules consisting of plasmidDNA sequences plus inserted DNAelements (pink here).
Such hybrid molecules are calledchimeric plasmids.
Figure 12.2 An EcoRI restrictionfragment of foreign DNA can beinserted into a plasmid.
Chimeric Plasmids
Named for mythological beasts with bodyparts from several creatures
After cleavage of a plasmid with a restriction enzyme, aforeign DNA fragment can be inserted
Ends of the plasmid/fragment are joined to form a"recombinant plasmid"
Recombinant plasmid can replicate when placed in asuitable bacterial host
See Figure 12.2
7/29/2019 Chem 4311 Chapter10 12 Nucleic Acid
31/36
Short DNA Duplexes With Restriction Sites Can BeUsed as Linkers
Figure 12.3 The use oflinkers to create tailor-madeends on cloning fragments.
Directional Cloning
Often one desires to insert foreign DNA in a particular
orientation This can be done by cleaving the plasmid with twodifferent restriction enzymes
Cleave the foreign DNA with same two restrictionenzymes
Foreign DNA can only be inserted in one direction
See Figure 12.4
Directional Cloning
Figure 12.4 Directional cloning.DNA molecules whose endshave different overhangs canbe used to form chimericconstructs in which the foreignDNA can enter the plasmid inonly one orientation.
Biologically Functional Chimeric Plasmids
Plasmids can be used to transform recipient E. colicells
( Transformation means the uptake and replicationof exogenous DNA by a recipient cell.)
To facilitate transformation, the bacterial cells arerendered somewhat permeable to DNA by Ca2+
treatment and a brief 42 C heat shock
The useful upper limit on cloned inserts in plasmidsis about 10 kbp. Many eukaryotic genes exceed thissize.
Biologically Functional Chimeric Plasmids
Figure 12.5 A typicalbacterial transformationexperiment. HerepBR322 is the cloningvector.
Shuttle Vectors Are Plasmids That Can Propagatein Two Different Organisms
Figure 12.6 A typical shuttle vector. LEU2+ is a gene in theyeast pathway for leucine biosynthesis.
Shuttle vectors are plasmids capable of propagating andtransferring ( shuttling ) genes between two different organisms.
7/29/2019 Chem 4311 Chapter10 12 Nucleic Acid
32/36
12.2 What Is a DNA Library?
A DNA library is a set of cloned DNA
fragments that together represent thegenes of a particular organism
Any particular gene may represent a tiny, tinyfraction of the DNA in a given cell
Can't isolate it directly
Trick is to find the fragment or fragments in thelibrary that contain the desired gene
12.2 What is a DNA Library?
The probabilities are daunting
Consider the formula on page 386 for probability of finding aparticular fragment in N clones
Suppose you want a 99% probability of finding a givenfragment in N clones of 10 kbp fragments
If your library is from the human genome, you would need1,400,000 clones to reach 99% probability of finding thefragment of interest!
Colony HybridizationA way to screen plasmid-based genome libraries for a DNA
fragment of interest
Host bacteria containing a plasmid-based library of DNAfragments are plated on a petri dish and allowed to growovernight to form colonies
Replica of colonies on the dish made with a nitrocellulose disc
Disc is treated with base or heated to convert dsDNA tossDNA and incubated with a labeled probe
Colonies that bind probe (labeled with 32P or other tag) holdthe fragment of interest
What is a DNA Library?
Figure 12.7 Screening a genomiclibrary by colony hybridization. Hostbacteria transformed with a plasmid-based genomic library are plated on apetri plate and incubated overnight toallow bacterial colonies to form.
A replica of the colonies is obtainedby overlaying the plate with a flexibledisc composed of absorbent material(such as nitrocellulose or nylon).
Probes for Southern Hybridization Can BePrepared in a Variety of Ways
Figure 12.8 Cloning genes usingoligonuceotide probes from a known aminoacid sequence. A radioactively labeled setof DNA (degenerate) oligonucleotidesrepresenting all possible mRNA codingsequences is synthesized and is used toprobe the genomic library by colonyhybridization (see Figure 12.7).
Labeling methodologies other thanradioactivity are also available.
Identifying Specific DNA Sequences by SouthernBlotting
Finding one particular DNA segment among a vast populationof different DNA fragments (e.g., in a genomic DNApreparation) is to exploit its sequence specificity to identify it.
Southern blots (invented by E.M. Southern) do this
DNA fragments (the library ) are fractionated by size withagarosegel electrophoresis
Separated molecules are blotted to an absorbent support andthen incubated with labeled (radioactive or otherwise)oligonucleotideprobes
Detection of the label shows the location of DNA fragmentsthat hybridized with the probe
7/29/2019 Chem 4311 Chapter10 12 Nucleic Acid
33/36
Identifying Specific DNA Sequences by SouthernBlotting
The Southern blottingtechnique involves thetransfer ofelectrophoreticallyseparated DNAfragments to anabsorbent sheet andsubsequent detection ofthe specific DNAsequences.
cDNA Libraries Are DNA Libraries Preparedfrom mRNA
cDNAs are DNAs copied from mRNA templates. cDNA libraries are constructed by synthesizing cDNA from
purified cellular mRNA.
Because most eukaryotic mRNAs carry 3'-poly(A) tails, mRNA canbe selectively isolated from preparations of total cellular RNA byoligo(dT)-cellulose chromatography (Figure 12.9)
DNA copies of the purified mRNAs are synthesized by firstannealing short oligo(dT) chains to the poly(A) tails.
These serve as primers for reverse transcriptase-driven synthesisof DNA (Figure 12.10)
cDNA Libraries Are DNA Libraries Preparedfrom mRNA
Figure 12.9 Isolation of eukaryotic mRNA viaoligo(dT)-cellulose chromatography.
cDNA Libraries Are DNA Libraries Preparedfrom mRNA
Reverse transcriptase is an enzyme that synthesizes a DNAstrand, copying RNA as the template
DNA polymerase is then used to copy the DNA strand andform a double-stranded duplex DNA
Linkers are then added to the DNA duplexes rendered fromthe mRNA templates
The cDNA is then cloned into a suitable vector
Once a cDNA derived from a particular gene has beenidentified, the cDNA becomes an effective probe for screeninggenomic libraries for isolation of the gene itself
cDNA Libraries Are DNA Libraries Preparedfrom mRNA
Figure 12.10 Reversetranscriptase-drivensynthesis of cDNA from
oligo(dT) primers annealedto the poly(A) tails ofpurified eukaryotic mRNA.
DNA Microarrays Are Arrays of DifferentOligonucleotides Immobilized on a Chip
Robotic methods can be used to synthesize combinatoriallibraries of DNA oligonucleotidesdirectly on a solid support.
The completed library is a 2-D array of differentoligonucleotides
The final products of such procedures are referred to as genechips because the sequences synthesized upon the chip
represent the sequences of chosen genes The oligonucleotideson such gene chips are used as probes in
hybridization experiments to reveal gene expression patterns
7/29/2019 Chem 4311 Chapter10 12 Nucleic Acid
34/36
DNA Microarrays Are Arrays of DifferentOligonucleotides Immobilized on a Chip
Figure 12.11 Gene chips (DNAmicroarrays) in the analysis ofgene expression.
The Human Genome Project
A working draft of thehuman genome wascompleted in June, 2000and published inFebruary 2001.
The genomes of manyother organisms havenow been sequenced aswell.
Information about wholegenome sequences hascreated a new branch ofscience calledbioinformatics.
12.3 Can the Cloned Genes in Libraries BeExpressed?
Figure 12.12 Expression vectorscarrying the promoter recognized bythe RNA polymerase of bacteriophageSP6 are useful for the production of
multiple RNA copies of any DNAinserted at the polylinker.
Expression vectors are engineeredso that the RNA or protein productsof cloned genes can be expressed.
12.3 Can the Cloned Genes in Libraries BeExpressed?
Figure 12.13 A typical expression-cloning vector.
To express a eukaryotic protein in E. coli, the eukaryoticcDNA must be cloned in an expression vector that containsregulatory signals for transcription and translation.
12.3 Can the Cloned Genes in Libraries BeExpressed?
Figure 12.14 A ptacprotein expressionvector contains thehybrid promoter ptacderived from fusionof the lac and trppromoters.
Strong promotershave beenconstructed to drivesynthesis of foreignproteins to levels of30% of total E. coliprotein.
12.3 Can the Cloned Genes in Libraries BeExpressed?
Figure 12.15 A typicalexpression vector forthe synthesis of ahybrid protein.
Some expressionvectors carry cDNAinserts cloned directlyinto the codingsequence of aprotein-coding gene.
7/29/2019 Chem 4311 Chapter10 12 Nucleic Acid
35/36
12.3 Can the Cloned Genes in Libraries BeExpressed? Reporter Gene Constructs
Figure 12.16 Greenfluorescent protein(GFP) as a reportergene.
Reporter gene
constructs arechimeric DNAmoleculescomposed of generegulatorysequences next toan easily
expressible geneproduct.
Specific Protein-Protein Interactions Can BeIdentified Using the Two-Hybrid System
Figure 12.17 The yeast two-hybrid system for identifyingprotein-protein interactions.If proteins X and Y interact,the lacZ reporter gene isexpressed. Cellsexpressing lacZ exhibit -galactosidase activity.
12.4 What Is the Polymerase Chain Reaction(PCR)?
What if you don't have enough DNA forcolony hybridization or Southern blots?
The small sample of DNA can serve as template for DNApolymerase
Make complementary primers; add DNA polymerase Add primers in more than 1000-fold excess Heat to separate dsDNA stran ds, then cool Run DNA polymerase (usually Taq) reaction again Repeat heating, cooling, polymerase cycle
12.4 What Is the Polymerase Chain Reaction(PCR)?
Figure 12.18 Polymerase chainreaction (PCR).
In Vitro MutagenesisFigure 12.19 One method of PCR-based site-directed mutagenesis.
(1) Template DNA strands areseparated and amplified byPCR.
(2) Following many cycles of PCR,the DNA product can be used totransform E. colicells.
(3) The plasmid DNA can beisolated and screened for thepresence of the uniquerestriction site (by restrictionendonuclease cleavage.
7/29/2019 Chem 4311 Chapter10 12 Nucleic Acid
36/36
12.5 How Is RNA Interference Used to Reveal theFunction of Genes?
RNA interference (RNAi) has emerged as amethod of choice in eukaryotic geneinactivation
RNAi leads to targeted destruction of aselected gene s transcript
The consequences following loss of genefunction reveal the role of the gene product incell metabolism
12.6 Is It Possible to Make Directed Changes inthe Heredity of an Organism?
Figure 12.20 Gene knockdown byRNAi.
The dsRNA is processed byDICER. Following unwinding byDICER, the guide strand isdelivered to the RISC complex.The guide strand and acomplementary mRNA arebrought together by Ago. RNaseon Ago cleaves the gene transcript(mRNA), rendering it incapable oftranslation by ribosomes.
Human Gene Therapy Can Repair GeneticDeficiencies
Figure 12.21 Retrovirus-mediatedgene delivery ex vivo using MMLV.
A basic strategy of human gene therapyinvolves incorporation of a functionalgene into target cells.Retroviruses (RNA viruses that makeDNA from RNA) provide a route forpermanent modification of host cells exvivo.
Human Gene Therapy Can Repair GeneticDeficiencies
Figure 12.22 Adenovirus-mediatedgene delivery in vivo.Adenoviruses are DNA viruses.
Adenovirus vectors are a possible invivo approach to human gene therapy.