CHEM 4311 General Biochemistry I Fall 2012

Chapter 10-12 Nucleotides and Nucleic Acids EXAM - October 19

Instructor: Office: Phone: email: Office hours Dr. Khairul I Ansari 316CPB 817-272-0616 kansari@uta.edu 12 am 1:30 pm Tuesday &.Thursday

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)

10.1 What Are the Structure and Chemistry of Nitrogenous 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.

10.1 What Are the Structure and Chemistry of Nitrogenous Bases?

Figure 10.3 The common pyrimidine bases cytosine, uracil, and thymine in the tautomeric forms predominant at pH 7.

10.1 What Are the Structure and Chemistry of Nitrogenous Bases?

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

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

Figure 10.6 The keto-enol tautomerism of uracil.

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

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

10.2 What Are Nucleosides?

Figure 10.9 The linear and cyclic (furanose) forms of ribose.

10.2 What Are Nucleosides?

Figure 10.9 The linear and cyclic (furanose) forms of deoxyribose.

10.2 What Are Nucleosides?

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

10.2 What Are Nucleosides?

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.

10.3 What Is the Structure and Chemistry of Nucleotides?

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.

10.3 What Is the Structure and Chemistry of Nucleotides?

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

Nucleoside 5'-Triphosphates Are Carriers of Chemical Energy

Figure 10.14 Phosphoryl, pyrophosphoryl, and nucleotidyl group transfer, the major biochemical reactions of nucleotides. Phosphoryl group transfer is shown here.

Nucleoside 5'-Triphosphates Are Carriers of Chemical Energy

Figure 10.14 Phosphoryl, pyrophosphoryl, and nucleotidyl group transfer, the major biochemical reactions of nucleotides. Pyrophosphoryl group transfer is shown here.

Nucleoside 5'-Triphosphates Are Carriers of Chemical Energy

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

10.4 What Are Nucleic Acids?

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.

10.4 What Are Nucleic Acids?

Figure 10.15 3,5phosphodiester 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

T
_____ _____

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

The Base Pairs Postulated by Watson

C
_____ _____ _____

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 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

Messenger RNA Carries the Sequence Information for Synthesis of a Protein

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.

Messenger RNA Carries the Sequence Information for Synthesis of a Protein

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 selfreplicating, 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 (5methyl-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

10.6 Are Nucleic Acids Susceptible to Hydrolysis?

Figure 10.27 Alkaline hydrolysis of RNA. Nucleophilic attach by OH- on the P atom leads to 5'-phosphoester cleavage.

10.6 Are Nucleic Acids Susceptible to Hydrolysis?

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.

10.6 Are Nucleic Acids Susceptible to Hydrolysis?

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 -----------GAATTC--------------------CAATTG----------

BamHI -----------GGATCC--------------------CCTAGG----------

MstI

-----------TGCGCA--------------------ACGCGT----------

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

11.1 How Do Scientists Determine the Primary Structure of Nucleic Acids?

Figure 11.1 DNA replication yields two daughter DNA duplexes identical to the parental DNA molecule.

11.1 How Do Scientists Determine the Primary Structure of Nucleic Acids?

Figure 11.2 Primed synthesis of a DNA template by DNA polymerase, using the four deoxynucleoside triphosphates as the substrates.

11.1 How Do Scientists Determine the Primary Structure of Nucleic Acids?

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.

Chain Termination Method

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

Chain Termination Method

Reaction mixtures can be separated by capillary electrophoresis Short fragments go to bottom, long fragments 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' on

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 + SO42Luciferase: 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 DoubleStranded 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 glycosidic bond 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 DoubleStranded 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 DoubleStranded 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 , 11 bp per turn B: right-handed, longer, thinner, 3.32 , 10 bp per turn Z: left-handed, longest, thinnest, 3.8 , 12 bp per turn See Figure 11.11

DNA Methylation and Epigenetics

Methylation of cytosine residues (forming 5methylcytosine) 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 , 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.

Nucleic Acid Hybridization: Different DNA Strands of Similar Sequence Can Form Hybrid Duplexes
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
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 bp per 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

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.

Figure 11.25 A model for the action of bacterial DNA gyrase (topoisomerase II).

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 DNAs 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.

Higher-Order Structural Organization of Chromatin Gives Rise to Chromosomes

Figure 11.30 A model for chromosome structure, human chromsome 4. The 30-nm fiber is created when the array of nucleosomes adopts a two-start helical conformation.

Higher-Order Structural Organization of Chromatin Gives Rise to Chromosomes

Figure 11.30 A model for chromosome structure, human chromsome 4. The 30 nm filament forms long DNA loops of variable length, each containing on average between 60,000 and 150,000 bp.

Higher-Order Structural Organization of Chromatin Gives Rise to Chromosomes

Figure 11.30 A model for chromosome structure, human 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.

Higher-Order Structural Organization of Chromatin Gives Rise to Chromosomes

Figure 11.30 A model for chromosome structure, human 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 12 Recombinant 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 42C 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
Shuttle vectors are plasmids capable of propagating and transferring (shuttling) genes between two different organisms.

Figure 12.6 A typical shuttle vector. LEU2+ is a gene in the yeast pathway for leucine biosynthesis.

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 Hybridization
A 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 plasmidbased 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)

cDNA Libraries Are DNA Libraries Prepared from mRNA

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

cDNA Libraries Are DNA Libraries Prepared from mRNA

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?

Expression vectors are engineered so that the RNA or protein products of cloned genes can 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.

12.3 Can the Cloned Genes in Libraries Be Expressed?

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 12.13 A typical expression-cloning vector.

12.3 Can the Cloned Genes in Libraries Be Expressed?

Strong promoters have been constructed to drive synthesis of foreign proteins to levels of 30% of total E. coli protein. Figure 12.14 A ptac protein expression vector contains the hybrid promoter ptac derived from fusion of the lac and trp promoters.

12.3 Can the Cloned Genes in Libraries Be Expressed?

Some expression vectors carry cDNA inserts cloned directly into the coding sequence of a protein-coding gene.

Figure 12.15 A typical expression vector for the synthesis of a hybrid protein.

Reporter Gene Constructs

Reporter gene constructs are chimeric DNA molecules composed of gene regulatory sequences next to an easily expressible gene product.

Figure 12.16 Green fluorescent protein (GFP) as a reporter gene.

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).