Chem 4311 Chapter10 12 Nucleic Acid Cropped

Chapter 10-12 Nucleotides and Nucleic Acids

EXAM - October 19

Instructor: Dr. Khairul I Ansari

Office: 316CPB

Phone: 817-272-0616

email: [email protected]

Office hours 12 am – 1:30 pm Tuesday &.Thursday

CHEM 4311

General Biochemistry I

Fall 2012

Chapter 10

Nucleotides and Nucleic Acids

“We have discovered the

secret of life.”Francis Crick, to patrons of

The Eagle, a pub in

Cambridge, England

(1953)

Francis Crick (right)

and James Watson

(left) point out features of

their model for the

structure of DNA.

Information Transfer in Cells

Figure 10.1 The

fundamental process of

information transfer in

cells.

10.1 What Are the Structure and Chemistry of Nitrogenous Bases?

Know the basic structures

• Pyrimidines

– Cytosine (DNA, RNA)

– Uracil (RNA)

– Thymine (DNA)

• Purines

– Adenine (DNA, RNA)

– Guanine (DNA, RNA)


Figure 10.2 (a) The pyrimidine ring system; by convention,

atoms are numbered as indicated.

(b) The purine ring system; atoms numbered as shown.


Figure 10.3 The common pyrimidine bases –

cytosine, uracil, and thymine – in the tautomeric forms

predominant at pH 7.


Figure 10.4 The common purine bases – adenine and

guanine – in the tautomeric forms predominant at pH 7.

The Properties of Pyrimidines and Purines Can Be Traced to Their Electron-Rich Nature

• The aromaticity and electron-rich nature of pyrimidines and purines enable them to undergo keto-enol tautomerism

• The keto tautomers of uracil, thymine, and guanine predominate at pH 7

• By contrast, the enol form of cytosine predominates at pH 7

• Protonation states of the nitrogens determines whether they can serve as H-bond donors or acceptors

• Aromaticity also accounts for strong absorption of UV light


Figure 10.6 The keto-enol tautomerism of uracil.


Figure 10.7 The tautomerization of the purine guanine.

Average UV absorbance nucleotides is ~ 260nM

10.2 What Are Nucleosides?

Structures to Know

• Nucleosides are compounds formed when a base is linked 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 and stability


Figure 10.9 The linear and cyclic (furanose) forms of

ribose.


Figure 10.9 The linear and cyclic (furanose) forms of

deoxyribose.


• The base is linked to the sugar 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


Figure 10.10 The common ribonucleosides.

Adenosine: A Nucleoside with Physiological Activity

• Adenosine functions as an

autacoid, or local hormone, and

neuromodulator.

• Circulating in the bloodstream, it

influences blood vessel dilation,

smooth muscle contraction,

neurotransmitter release, and fat

metabolism.

• Adenosine is also a sleep

regulator. Adenosine rises during

wakefulness, promoting eventual

sleepiness.

• Caffeine promotes wakefulness

by blocking binding of adenosine

to its neuronal receptors.

10.3 What Is the Structure and Chemistry of Nucleotides?

Figure 10.11 Structures of the four common ribonucleotides –

AMP, GMP, CMP, and UMP. Also shown: 3’-AMP.


Figure 10.13 Formation of ADP and ATP by the succesive

addition of phosphate groups via phosphoric anhydride

linkages. Note that the reaction is a dehydration synthesis.


Figure 10.13 Formation of ADP and ATP by the succesive

addition of phosphate groups via phosphoric anhydride

linkages. Note that the reaction is a dehydration synthesis.

Nucleoside 5'-Triphosphates Are Carriers of Chemical Energy

• Nucleoside 5'-triphosphates are indispensable agents in metabolism because their phosphoric anhydride bonds are a source of chemical energy

• Bases serve as recognition units • ATP is central to energy metabolism • GTP drives protein synthesis • CTP drives lipid synthesis • UTP drives carbohydrate metabolism


Figure 10.14 Phosphoryl, pyrophosphoryl, and nucleotidyl

group transfer, the major biochemical reactions of

nucleotides. Phosphoryl group transfer is shown here.


Figure 10.14 Phosphoryl, pyrophosphoryl, and

nucleotidyl group transfer, the major biochemical

reactions of nucleotides. Pyrophosphoryl group

transfer is shown here.


Figure 10.14 Phosphoryl, pyrophosphoryl, and

nucleotidyl group transfer, the major biochemical

reactions of nucleotides. Nucleotidyl group transfer is

shown here.

10.4 What Are Nucleic Acids?

• Nucleic acids are linear polymers of nucleotides

linked 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


Figure 10.15 3',5'-

Phosphodiester bridges link

nucleotides together to form

polynucleotide chains. The 5'-

ends of the chains are at the

top; the 3'-ends are at the

bottom. RNA is shown here.


Figure 10.15 3’,5’-phosphodiester bridges link

nucleotides together to form

polynucleotide chains. The

5’-ends of the chains are at

the top; the 3’-ends are at the bottom. DNA is shown

here.

10.5 What Are the Different Classes of Nucleic Acids?

• DNA - one type, one purpose

• RNA - 3 (or 4) types, 3 (or 4) purposes

– ribosomal RNA - the basis of structure and function of ribosomes

– messenger RNA - carries the message for protein synthesis

– transfer RNA - carries the amino acids for protein synthesis

– Others:• Small nuclear RNA

• Small non-coding RNAs

10.5 What Are the Different Classes of Nucleic Acids?

Figure 10.16 The

antiparallel nature of the

DNA double helix. The two

chains have opposite

orientations.

The DNA Double Helix

The double helix is stabilized by hydrogen bonds

• "Base pairs" arise from hydrogen bonds A-T; G-C

• Erwin Chargaff had the pairing data, but didn't

understand its implications

• Rosalind Franklin's X-ray fiber diffraction data

was 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.

AT_____

_____

The Base Pairs Postulated by Watson

Figure 10.17 The Watson-Crick base pairs A:T and G:C.

G:C is shown here.

GC_____

_____

_____

The Structure of DNA

An antiparallel double helix

• Diameter of 2 nm

• Eukaryotic DNA wrapped around histone

proteins to form nucleosomes

• Base pairs: A-T, G-C

• Total Length of human DNA is ~ 3.3 billion bp

The Structure of DNA

Figure 10.18 Replication of DNA

gives identical progeny molecules

because base pairing is the

mechanism that determines the

nucleotide sequence of each newly

synthesized strand.

Messenger RNA Carries the Sequence Information for

Synthesis of a Protein

Transcription product of DNA

• In prokaryotes, a single mRNA contains the information for synthesis of many proteins

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



Figure 10.20 Transcription and translation of mRNA

molecules in prokaryotic versus eukaryotic cells.

In prokaryotes, a single mRNA molecule may contain the

information for the synthesis of several polypeptide

chains within its nucleotide sequence.



Figure 10.20 Transcription and translation of mRNA molecules

in prokaryotic versus eukaryotic cells.

Eukaryotic mRNAs encode only one polypeptide but are more

complex.

Eukaryotic mRNA

• DNA is transcribed to produce heterogeneous nuclear 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 and Functional

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 acid sequence of a polypeptide chain by ribosomes

Ribosomal RNA Provides the Structural and Functional

Foundation for Ribosomes

Figure 10.22 The organization and composition of ribosomes.

Transfer RNAs Carry Amino Acids to Ribosomes for 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

The RNA World and Early Evolution

• Thomas Cech and Sidney Altman showed that RNA molecules are not only informational – they can also be catalytic

• This gave evidence to the postulate by Francis Crick and 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

Glycolaldehyde has been detected at the

center of the Milky Way and could be a

precursor of ribose and glucose.

The Chemical Differences Between DNA and RNA Have

Biological Significance

• Two fundamental chemical differences distinguish DNA from RNA:

– DNA contains 2-deoxyribose instead of ribose

– DNA contains thymine instead of uracil

The Chemical Differences Between DNA and RNA Have

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 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

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


Figure 10.27 Alkaline hydrolysis of RNA. Nucleophilic

attach by OH- on the P atom leads to 5'-phosphoester

cleavage.


Figure 10.27 Alkaline hydrolysis of RNA. Nucleophilic attack by

OH- on the P atom leads to 5'-phosphoester cleavage. Random

hydrolysis of the cyclic phosphodiester intermediate gives a

mixture of 2'- and 3'-nucleoside monophosphate products.


Figure 10.28 Cleavage in polynucleotide chains. Cleavage

on the a side leaves the phosphate attached to the 5'-

position of the adjacent nucleotide. b-side hydrolysis yields

3'-phosphate products.

Restriction Enzymes

• Bacteria have learned to "restrict" the possibility of attack from foreign DNA by means of "restriction enzymes"

• Type II and III restriction enzymes cleave DNA chains at selected sites

• Enzymes may recognize 4, 6 or more bases in selecting sites for cleavage

• An enzyme that recognizes a 6-base sequence is a "six-cutter"

Type II Restriction Enzymes

• No ATP requirement

• Recognition sites in dsDNA 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 - species

• Following letter denotes strain

• EcoRI is the first restriction enzyme isolated from the R strain of E. coli

Cleavage Sequences of Restriction Endonucleases

EcoRI -----------G↓AATTC----------

-----------CAATT↑G----------

BamHI -----------G↓GATCC----------

-----------CCTAG↑G----------

MstI -----------TGC↓GCA----------

-----------ACG↑CGT----------

Chapter 11

Structure of Nucleic Acids

11.1 How Do Scientists Determine the Primary

Structure of Nucleic Acids?

• Two simple tools have made nucleic acid sequencing easier than polypeptide sequencing:

– The type II restriction endonucleases that cleave DNA at specific oligonucleotide sites

– Gel electrophoresis, which is capable of separating nucleic acid fragments that differ from one another in length by just a single nucleotide

11.1 How Do Scientists Determine the

Primary Structure of Nucleic Acids?

• Chain termination method (dideoxy method), developed by Frederick Sanger is the basis for most DNA sequencing currently.

• The method takes advantage of the DNA polymerase reaction, which copies a DNA strand in complementary fashion to form a new second strand



Figure 11.1 DNA replication yields two

daughter DNA duplexes identical to the

parental DNA molecule.



Figure 11.2 Primed synthesis of a DNA template by

DNA polymerase, using the four deoxynucleoside

triphosphates as the substrates.



• DNA is a double-helical molecule • Each strand of the helix must be copied in

complementary 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 with a complementary primer is copied by DNA polymerase in the presence of dATP, dCTP, dGTP, dTTP

• Solution contains small amounts of the four dideoxynucleotide analogs of these substrates, each of 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 dideoxynucleotide

terminates further synthesis of that strand

Figure 11.3 The chain

termination method of

DNA sequencing.


• Most of the time, the polymerase uses normal nucleotides and DNA molecules grow normally

• Occasionally, the polymerase uses a dideoxynucleotide, which prevents further extension when added to the growing chain

• Random insertion of dd-nucleotides leaves (optimally) at least a few chains terminated at every occurrence of a given nucleotide


• Reaction mixtures can be separated by capillary electrophoresis

• 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'

The set of terminated strands can be separated

by capillary electrophoresis

High-Throughput DNA Sequencing by the Light of

Fireflies

• The importance of DNA sequence information has motivated development of more rapid and efficient DNA sequencing technologies

• Multiple copies of template DNA molecules are immobilized on microscopic beads

• Reagents for primed synthesis are passed over the beads

• Pyrophosphate release is monitored by light emission via ATP sulfurylase and luciferase reactions

High-Throughput DNA Sequencing by the Light of

Fireflies

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

Emerging Technologies to Sequence DNA are Based on

Single-Molecule Sequencing Strategies

• Growing demand for sequence information is driving the development of faster and cheaper methods of DNA sequencing

• One technique involves passing a single strand of DNA through a graphene monolayer pore, measuring the change in electrical conductance (ion flow) through the pore

• Each base alters electrical conductance in a subtle but different way, facilitating the “reading” of sequence

Figure 11.5 DNA Sequencing through a pore in a graphene monolayer

11.2 What Sorts of Secondary Structures Can Double-

Stranded DNA Molecules Adopt?

• Polynucleotide strands are flexible

• Each deoxyribose-phosphate segment of the backbone has six degrees of freedom (Fig 11.4a)

• Furanose rings are not planar but instead adopt puckered conformations, four of which are shown in Figure 11.4b

• A seventh degree of freedom per nucleotide arises because of free rotation about the C1'-N glycosidicbond

• This freedom allows the plane of the base to rotate relative 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 the

deoxyribose-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 furanose

rings.

Figure 11.7

(a) Double-stranded DNA as an imaginary

ladderlike structure.

(b) A simple right-handed twist converts the

ladder 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 of phosphate groups, which makes them most stable on the helix exterior

• Base-pair stacking interactions

• Right-twist closes the gaps between base pairs to 3.4 A (0.34 nm) in B-DNA

The ““““canonical”””” base pairs

• 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 backbone also form H bonds

Major and minor grooves• The "tops" of the bases (as we draw

them) line the "floor" of the major groove

• The major groove is large enough to accommodate an alpha helix from a protein

• Regulatory proteins (transcription factors) can recognize the pattern of bases and the H-bonding possibilities in the major groove

Double Helical Structures Can Adopt a Number of Stable Conformations

Figure 11.11 The B-form of the

DNA double helix. In B-form, the

pitch (the distance required to

complete one helical turn) is 3.4

nm. Twelve base pairs of DNA are

shown.

Comparison of A, B, Z DNA

See Table 11.1

• A: right-handed, short and broad, 2.3 Å, 11 bp per turn

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

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

• See Figure 11.11

DNA Methylation and Epigenetics

• Methylation of cytosine residues (forming 5-methylcytosine) is essential for normal embryonic development

• Cytosine methylation switches genes off, so that the information they encode is not expressed

• Epigenetics is the study of heritable changes in the genome that occur without a change in nucleotide sequence (such as cytosine methylation)

• Epigenetic changes can influence expression of the information 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 of fused, heterocyclic rings, can slip between the stacked pairs of bases

• The bases are force apart to accommodate these intercalating agents

– Ethidium bromide

– Acridine orange

– Actinomycin D

11.3 Can the Secondary Structure of DNA Be

Denatured and Renatured?

• 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 π,π electron interactions

• When T is lowered, the absorbance drops, reflecting re-establishment of the double helix and base-pair stacking

11.3 Can the Secondary Structure of DNA Be

Denatured and Renatured?

Figure 11.18 Heat denaturation of DNA from various sources.

Single-Stranded DNA Can Renature to Form DNA

Duplexes

• Denatured DNA will renature to re-form the duplex structure if the denaturing conditions are removed

• Renaturation requires reassociation of the DNA strands into a double helix, a process termed reannealing

• For this to occur, the strands must realign so that their complementary bases are once again in register and the helix can be “zippered up”

Single-Stranded DNA Can Renature to Form DNA

Duplexes

Figure 11.21 Steps in the

thermal denaturation and

renaturation of DNA.

• If DNA from two different species are mixed, denatured, and allowed to cool slowly, hybrid duplexes may form, provided the DNA from one species is similar in sequence to the other

• The degree of hybridization is a measure of the sequence similarity between the two species

• 25% of the DNA from a human forms hybrids with mouse DNA, implying some sequence similarity

• Hybridization is a common procedure in molecular biology for identifying specific genes and for revealing evolutionary relationships

Nucleic Acid Hybridization: Different DNA Strands of

Similar Sequence Can Form Hybrid Duplexes

Nucleic Acid Hybridization: Different DNA Strands of Similar

Sequence Can Form Hybrid Duplexes

Figure 11.22 Solutions of

human DNA (red) and mouse

DNA (blue) are mixed and

denatured; then, the single

strands are allowed to

reanneal.

About 25% of human DNA

forms hybrid duplexes with

mouse DNA.

11.4 Can DNA Adopt Structures of Higher

Complexity?

• 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 can introduce or remove supercoils

• Cruciforms occur in palindromic regions of DNA

• Negative supercoiling may promote cruciforms

Supercoils Are One Kind of Structural

Complexity in DNA

Double-stranded circular DNA forms supercoils, if the strands 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)

DNA Gyrase is a topoisomerase that introduces

negative supercoils into DNA

Figure 11.25 A model for the action of

bacterial DNA gyrase (topoisomerase II).

Negative supercoils cause a torsional

stress on the molecule, so the

molecule tends to unwind. Negative

supercoiling makes it easier to

separate DNA strands and access the

information encoded by the sequence.

DNA Gyrase is a topoisomerase that introduces

negative supercoils into DNA

Figure 11.25 Conformational

changes in the enzyme allow an

intact region of the DNA duplex

to pass between the cut ends.

The cut ends are religated (3),

and the covalently complete

DNA duplex is released from the

enzyme. The circular DNA now

contains two negative

supercoils (4).

11.5 What Is the Structure of Eukaryotic

Chromosomes?

• 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 than 100,000!

• It is made possible by wrapping the DNA around protein spools called nucleosomes and then packing these in helical filaments

• These filaments are thought to arrange in loops associated with the nuclear matrix

Nucleosomes Are the Fundamental Structural Unit

in Chromatin

Histones and nonhistone chromosomal proteins are the two

classes of chromatin proteins. Five distinct histones are

known: H1, H2A, H2B, H3, and H4.

Pairs of histones H2A, H2B, H3, and H4 aggregate to

form octameric core structures; the DNA helix is wound

around these core octamers, creating nucleosomes.

Nucleosome Structure

• Chromatin, the nucleoprotein complex, consists of histones and nonhistone chromosomal proteins

• Histone octamer structure has been solved (without DNA by Moudrianakis, and with DNA by Richmond)

• Nonhistone proteins are regulators of gene expression

The Structure of the Nucleosome – a Histone

Octamer wrapped with DNA

Figure 11.28

Structural Organization of Chromatin Gives Rise to

Chromosomes

• The beads-on-a-string motif is the “primary” structure of chromatin.

• The “secondary” level of chromatin structure is the 30-nm fiber, formed when an array of nucleosomes in a zig-zag pattern adopts a two-start helical conformation (Figures 11.29a, b, c).

• Higher levels of chromatin structural organization are achieved when the 30-nm fiber forms long loops of 60-150,000 bp.

• Electron microscopic analysis of human chromosome 4 suggests that 18 such loops are then arranged radially about the circumference of a single turn to form a miniband unit of the chromosome.

Higher-Order Structural Organization of Chromatin Gives Rise to Chromosomes

Figure 11.30 A model for chromosome structure, human

chromsome 4, showing nucleosomes in the “beads on a

string” motif.



chromsome 4. The 30-nm fiber is created when the array

of nucleosomes adopts a two-start helical conformation.



chromsome 4. The 30 nm filament forms long DNA loops of

variable length, each containing on average between

60,000 and 150,000 bp.



chromsome 4. Electron microscopic analysis of

chromosome 4 suggests that 18 loops are arranged

radially about the circumference of a single turn to form a

miniband unit of the chromosome.



chromsome 4. Approximately a million minibands are

arranged along a central axis in each of the chromatids of

chromosome 4 that form at mitosis.

SMC Proteins Aid Chromosome Organization and Mediate Chromosome Dynamics

Figure 11.31 SMC protein architecture and function. SMC

proteins range from 115 to 165 kD in size.

11.6 Can Nucleic Acids Be Synthesized Chemically?

• 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 of these constraints.

11.7 What Are the Secondary and Tertiary Structures of RNA?

• A number of defined structural motifs recur within the loops of stem-loop structures, such as U-turns, tetraloops, and bulges

• Regions where several stem-loop structures meet are termed junctions

• Stems, loops, bulges, and junctions are the four basic secondary structural elements in RNA

• Other tertiary structural motifs arise from coaxial

stacking, pseudoknot formation, and ribose zippers

Transfer RNA Adopts Higher-Order Structure Through Intrastrand Base Pairing

• In tRNA, with 73-94 nucleotides in a single chain, a majority of the bases are hydrogen- bonded to one another

• Hairpin turns bring complementary stretches of bases into contact

• Extensive H-bonding creates four double helical domains, 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 Through Intrastrand Base Pairing

Figure 11.36 A

general diagram for

the structure of tRNA.

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 leads to extensive base-pairing

• Secondary structure features seem to be conserved, whereas sequence is not

• There must be common designs and functions that must be conserved

Chapter 12Recombinant DNA: Cloning and

Creation of Chimeric Genes

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 linking new DNA fragments to the sticky ends of plasmid

• These recombinant molecules can be autonomously 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 foreign DNA will not disrupt replication or inactivate essential markers)

Plasmids as Cloning Vectors

Figure 12.1 One of

the first widely used

cloning vectors was

the plasmid pBR322.

Note the antibiotic

resistance genes

(ampr and tetr).

Virtually Any DNA Sequence Can Be Cloned

Nuclease cleavage at a restriction site

linearizes the circular plasmid so that a

foreign DNA fragment can be inserted.

Recombinant plasmids are hybrid

DNA molecules consisting of plasmid

DNA sequences plus inserted DNA

elements (pink here).

Such hybrid molecules are called

chimeric plasmids.

Figure 12.2 An EcoRI restriction

fragment of foreign DNA can be

inserted into a plasmid.

Chimeric Plasmids

Named for mythological beasts with body parts from several creatures

• After cleavage of a plasmid with a restriction enzyme, a foreign DNA fragment can be inserted

• Ends of the plasmid/fragment are joined to form a "recombinant plasmid"

• Recombinant plasmid can replicate when placed in a suitable bacterial host

Short DNA Duplexes With Restriction Sites Can Be Used as Linkers

Figure 12.3 The use of

linkers to create tailor-made

ends 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 two different restriction enzymes

• Cleave the foreign DNA with same two restriction enzymes

• Foreign DNA can only be inserted in one direction

Directional Cloning

Figure 12.4 Directional cloning.

DNA molecules whose ends

have different overhangs can

be used to form chimeric

constructs in which the foreign

DNA can enter the plasmid in

only one orientation.

Biologically Functional Chimeric Plasmids

• Plasmids can be used to transform recipient E. coli

cells

• (“Transformation” means the uptake and replication of exogenous DNA by a recipient cell.)

• To facilitate transformation, the bacterial cells are rendered somewhat permeable to DNA by Ca2+

treatment and a brief 42°C heat shock

• The useful upper limit on cloned inserts in plasmids is about 10 kbp. Many eukaryotic genes exceed this size.

Biologically Functional Chimeric Plasmids

Figure 12.5 A typical

bacterial transformation

experiment. Here

pBR322 is the cloning

vector.

Shuttle Vectors Are Plasmids That Can Propagate in Two Different Organisms

Figure 12.6 A typical shuttle vector. LEU2+ is a gene in the

yeast pathway for leucine biosynthesis.

Shuttle vectors are plasmids capable of propagating and

transferring (“shuttling”) genes between two different organisms.

12.2 What Is a DNA Library?

A DNA library is a set of cloned DNA

fragments that together represent the

genes of a particular organism

• Any particular gene may represent a tiny, tiny fraction of the DNA in a given cell

• Can't isolate it directly

• Trick is to find the fragment or fragments in the library 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 a particular fragment in N clones

• Suppose you want a 99% probability of finding a given fragment in N clones of 10 kbp fragments

• If your library is from the human genome, you would need 1,400,000 clones to reach 99% probability of finding the fragment 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 DNA fragments are plated on a petri dish and allowed to grow overnight to form colonies

• Replica of colonies on the dish made with a nitrocellulose disc

• Disc is treated with base or heated to convert dsDNA to ssDNA and incubated with a labeled probe

• Colonies that bind probe (labeled with 32P or other tag) hold the fragment of interest

What is a DNA Library?

Figure 12.7 Screening a genomic

library by colony hybridization. Host

bacteria transformed with a plasmid-

based genomic library are plated on a

petri plate and incubated overnight to

allow bacterial colonies to form.

A replica of the colonies is obtained

by overlaying the plate with a flexible

disc composed of absorbent material

(such as nitrocellulose or nylon).

Probes for Southern Hybridization Can Be Prepared in a Variety of Ways

Figure 12.8 Cloning genes using

oligonuceotide probes from a known amino

acid sequence. A radioactively labeled set

of DNA (degenerate) oligonucleotides

representing all possible mRNA coding

sequences is synthesized and is used to

probe the genomic library by colony

hybridization (see Figure 12.7).

Labeling methodologies other than

radioactivity are also available.

Identifying Specific DNA Sequences by Southern Blotting

• Finding one particular DNA segment among a vast population of different DNA fragments (e.g., in a genomic DNA preparation) 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 with agarose gel electrophoresis

• Separated molecules are blotted to an absorbent support and then incubated with labeled (radioactive or otherwise) oligonucleotide probes

• Detection of the label shows the location of DNA fragments that hybridized with the probe

Identifying Specific DNA Sequences by Southern Blotting

The Southern blotting

technique involves the

transfer of

electrophoretically

separated DNA

fragments to an

absorbent sheet and

subsequent detection of

the specific DNA

sequences.

cDNA Libraries Are DNA Libraries Prepared from 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 can be selectively isolated from preparations of total cellular RNA by oligo(dT)-cellulose chromatography (Figure 12.9)

• DNA copies of the purified mRNAs are synthesized by first annealing short oligo(dT) chains to the poly(A) tails.

• These serve as primers for reverse transcriptase-driven synthesis of DNA (Figure 12.10)


• Reverse transcriptase is an enzyme that synthesizes a DNA strand, copying RNA as the template

• DNA polymerase is then used to copy the DNA strand and form a double-stranded duplex DNA

• Linkers are then added to the DNA duplexes rendered from the mRNA templates

• The cDNA is then cloned into a suitable vector

• Once a cDNA derived from a particular gene has been identified, the cDNA becomes an effective probe for screening genomic libraries for isolation of the gene itself


Figure 12.10 Reverse

transcriptase-driven

synthesis of cDNA from

oligo(dT) primers annealed

to the poly(A) tails of

purified eukaryotic mRNA.

DNA Microarrays Are Arrays of Different Oligonucleotides Immobilized on a Chip

• Robotic methods can be used to synthesize combinatorial libraries of DNA oligonucleotides directly on a solid support.

• The completed library is a 2-D array of different oligonucleotides

• The final products of such procedures are referred to as “gene chips” because the sequences synthesized upon the chip represent the sequences of chosen genes

• The oligonucleotides on such gene chips are used as probes in hybridization experiments to reveal gene expression patterns

DNA Microarrays Are Arrays of Different Oligonucleotides Immobilized on a Chip

Figure 12.11 Gene chips (DNA

microarrays) in the analysis of

gene expression.

12.3 Can the Cloned Genes in Libraries Be Expressed?

Figure 12.12 Expression vectors

carrying the promoter recognized by

the RNA polymerase of bacteriophage

SP6 are useful for the production of

multiple RNA copies of any DNA

inserted at the polylinker.

Expression vectors are engineered

so that the RNA or protein products

of cloned genes can be expressed.


Figure 12.13 A typical expression-cloning vector.

To express a eukaryotic protein in E. coli, the eukaryotic

cDNA must be cloned in an expression vector that contains

regulatory signals for transcription and translation.


Figure 12.14 A ptac

protein expression

vector contains the

hybrid promoter ptac

derived from fusion

of the lac and trp

promoters.

Strong promoters

have been

constructed to drive

synthesis of foreign

proteins to levels of

30% of total E. coli

protein.


Figure 12.15 A typical

expression vector for

the synthesis of a

hybrid protein.

Some expression

vectors carry cDNA

inserts cloned directly

into the coding

sequence of a

protein-coding gene.

Reporter Gene Constructs

Figure 12.16 Green

fluorescent protein

(GFP) as a reporter

gene.

Reporter gene

constructs are

chimeric DNA

molecules

composed of gene

regulatory

sequences next to

an easily

expressible gene

product.

12.4 What Is the Polymerase Chain Reaction (PCR)?

What if you don't have enough DNA for

colony hybridization or Southern blots?

• The small sample of DNA can serve as template for DNA polymerase

• 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 chain

reaction (PCR).

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