Preface

Acknowledgments

I

FOUNDATIONS

1

Evolution, Science, and Molecular Biology

MOMENT OF DISCOVERY Jack Szostak, on his discovery of self-dividing vesicles that mimic growing cells

1.1 The Evolution of Life on Earth

What Is Life?

Evolution Underpins Molecular Biology

HIGHLIGHT 1-1 EVOLUTION Observing Evolution in the Laboratory

Life on Earth Probably Began with RNA

The Last Universal Common Ancestor Is the Root of the Tree of Life

Evolution by Natural Selection Requires Variation and Competition

1.2 How Scientists Do Science

Science Is a Path to Understanding the Natural Universe

The Scientific Method Underlies Scientific Progress

The Scientific Method Is a Versatile Instrument of Discovery

Scientists Work within a Community of Scholars

HOW WE KNOW

Adenine Could Be Synthesized with Prebiotic Chemistry

Clay Had a Role in Prebiotic Evolution

Darwin’s World Helped Him Connect the Dots

2

DNA: The Repository of Biological Information

MOMENT OF DISCOVERY James Berger, on his discovery of the structure and mechanism of topoisomerase II

2.1 Mendelian Genetics

Mendel’s First Law: Allele Pairs Segregate during Gamete Formation

Mendel’s Second Law: Different Genes Assort Independently during Gamete Formation

There Are Exceptions to Mendel’s Laws

2.2 Cytogenetics: Chromosome Movements during Mitosis and Meiosis

Cells Contain Chromosomes and Other Internal Structures

Mitosis: Cells Evenly Divide Chromosomes between New Cells

Meiosis: Chromosome Number Is Halved during Gamete Formation

2.3 The Chromosome Theory of Inheritance

Sex-Linked Genes in the Fruit Fly Reveal That Genes Are on Chromosomes

Linked Genes Do Not Segregate Independently

Recombination Unlinks Alleles

Recombination Frequency Can Be Used to Map Genes along Chromosomes

2.4 Foundations of Molecular Genetics

DNA Is the Chemical of Heredity

Genes Encode Polypeptides and Functional RNAs

The Central Dogma: Information Flows from DNA to RNA to Protein—Usually

Mutations in DNA Give Rise to Phenotypic Change

HIGHLIGHT 2-1 MEDICINE The Molecular Biology of Sickle-Cell Anemia, a Recessive Genetic Disease of Hemoglobin

HOW WE KNOW

Chromosome Pairs Segregate during Gamete Formation in a Way That Mirrors the Mendelian Behavior of Genes

Corn Crosses Uncover the Molecular Mechanism of Crossing Over

Hershey and Chase Settle the Matter: DNA Is the Genetic Material

3

Chemical Basis of Information Molecules

MOMENT OF DISCOVERY Roxana Georgescu, on her discovery of how beta processivity clamps bind DNA

3.1 Chemical Building Blocks of Nucleic Acids and Proteins

Nucleic Acids Are Long Chains of Nucleotides

Proteins Are Long Polymers of Amino Acids

Chemical Composition Helps Determine Nucleic Acid and Protein Structure

Chemical Composition Can Be Altered by Postsynthetic Changes

3.2 Chemical Bonds

Electrons Are Shared in Covalent Bonds and Transferred in Ionic Bonds

Chemical Bonds Are Explainable in Quantum Mechanical Terms

Forming and Breaking of Chemical Bonds Involves Energy Transfer

Electron Distribution between Bonded Atoms Determines Molecular Behavior

3.3 Weak Chemical Interactions

Van der Waals Forces Are Nonspecific Contacts between Atoms

The Hydrophobic Effect Brings Together Nonpolar Molecules

Adjacent Bases in Nucleic Acids Participate in Noncovalent Interactions

Hydrogen Bonds Are a Special Kind of Noncovalent Bond

Combined Effects of Weak Chemical Interactions Stabilize Macromolecular Structures

Weak Chemical Bonds Also Facilitate Macromolecular Interactions

3.4 Stereochemistry

Three-Dimensional Atomic Arrangements Define Molecules

Biological Molecules and Processes Selectively Use One Stereoisomer

Proteins and Nucleic Acids Are Chiral

HIGHLIGHT 3-1 MEDICINE The Behavior of a Peptide Made of d-Amino Acids

3.5 The Role of pH and Ionization

The Hydrogen Ion Concentration of a Solution Is Measured by pH

Buffers Prevent Dramatic Changes in pH

The Henderson-Hasselbalch Equation Estimates the pH of a Buffered Solution

3.6 Chemical Reactions in Biology

The Mechanism and Speed of Chemical Transformation Define Chemical Reactions

Biological Systems Follow the Laws of Thermodynamics

Catalysts Increase the Rates of Biological Reactions

Energy Is Stored and Released by Making and Breaking Phosphodiester Bonds

HIGHLIGHT 3-2 EVOLUTION ATP: The Critical Molecule of Energy Exchange in All Cells

HOW WE KNOW

Single Hydrogen Atoms Are Speed Bumps in Enzyme-Catalyzed Reactions

Peptide Bonds Are (Mostly) Flat

4

Protein Structure

MOMENT OF DISCOVERY Steve Mayo, on his discovery of the first successful method for computational protein design

4.1 Primary Structure

Amino Acids Are Categorized by Chemical Properties

Amino Acids Are Connected in a Polypeptide Chain

Evolutionary Relationships Can Be Determined from Primary Sequence Comparisons

HIGHLIGHT 4-1 A CLOSER LOOK Purification of Proteins by Column Chromatography and SDS-PAGE

4.2 Secondary Structure

The α Helix Is a Common Form of Secondary Protein Structure

The β Conformation Forms Sheetlike Structures

Reverse Turns Allow Secondary Structures to Fold

4.3 Tertiary and Quaternary Structures

Tertiary and Quaternary Structures Can Be Represented in Different Ways

Domains Are Independent Folding Units within the Protein

Supersecondary Structural Elements Are Building Blocks of Domains

Quaternary Structures Range from Simple to Complex

Intrinsically Unstructured Proteins Have Versatile Binding Properties

Protein Structures Help Explain Protein Evolution

HIGHLIGHT 4-2 A CLOSER LOOK Protein Structure Databases

4.4 Protein Folding

Predicting Protein Folding Is a Goal of Computational Biology

Polypeptides Fold through a Molten Globule Intermediate

HIGHLIGHT 4-3 MEDICINE Prion-Based Misfolding Diseases

Chaperones and Chaperonins Can Facilitate Protein Folding

Protein Isomerases Assist in the Folding of Some Proteins

4.5 Determining the Atomic Structure of Proteins

Most Protein Structures Are Solved by X-Ray Crystallography

Smaller Protein Structures Can Be Determined by NMR

HOW WE KNOW

Sequence Comparisons Yield an Evolutionary Roadmap from Bird Influenza to a Deadly Human Pandemic

We Can Tell That a Protein Binds ATP by Looking at Its Sequence

Disulfide Bonds Act as Molecular Cross-Braces to Stabilize a Protein

5

Protein Function

MOMENT OF DISCOVERY Smita Patel, on her early work with the T7 gene 4–encoded DNA helicase

5.1 Protein-Ligand Interactions

Reversible Binding of Proteins to Other Molecules Follows Defined Principles

Protein-Ligand Interactions Can Be Quantified

DNA-Binding Proteins Guide Genome Structure and Function

5.2 Enzymes: The Reaction Catalysts of Biological Systems

Enzymes Catalyze Specific Biological Reactions

Enzymes Increase the Rate of a Reaction by Lowering the Activation Energy

The Rates of Enzyme-Catalyzed Reactions Can Be Quantified

HIGHLIGHT 5-1 A CLOSER LOOK Reversible and Irreversible Inhibition

DNA Ligase Activity Illustrates Some Principles of Catalysis

5.3 Motor Proteins

Helicases Abound in DNA and RNA Metabolism

Helicase Mechanisms Have Characteristic Molecular Parameters

5.4 The Regulation of Protein Function

Modulator Binding Causes Conformational Changes in Allosteric Proteins

Allosteric Enzymes Have Distinctive Binding and/or Kinetic Properties

Autoinhibition Can Affect Enzyme Activity

Some Proteins Are Regulated by Reversible Covalent Modification

Phosphoryl Groups Affect the Structure and Catalytic Activity of Proteins

Some Proteins Are Regulated by Proteolytic Cleavage

HIGHLIGHT 5-2 Medicine HIV Protease: Rational Drug Design Using Protein Structure

HOW WE KNOW

The Discovery of the Lactose Repressor: One of the Great Sagas of Molecular Biology

The lacI Gene Encodes a Repressor

Discovery of the Lactose Repressor Helped Give Rise to DNA Sequencing

II

NUCLEIC ACID STRUCTURE AND METHODS  

6

DNA and RNA Structure

MOMENT OF DISCOVERY Jamie Cate, on determining the molecular structure of the bacterial ribosome

6.1 The Structure and Properties of Nucleotides

Nucleotides Comprise Phosphates and Characteristic Bases and Sugars

Phosphodiester Bonds Link the Nucleotide Units in Nucleic Acids

The Properties of Nucleotide Bases Affect the Three-Dimensional Structure of Nucleic Acids

Nucleotides Play Additional Roles in Cells

6.2 DNA Structure

DNA Molecules Have Distinctive Base Compositions

DNA Is Usually a Right-Handed Double Helix

DNA Adopts Different Helical Forms

Certain DNA Sequences Adopt Unusual Structures

HIGHLIGHT 6-1 TECHNOLOGY DNA Nanotechnology

6.3 RNA Structure

RNAs Have Helical Secondary Structures

RNAs Form Various Stable Three-Dimensional Structures

6.4 Chemical and Thermodynamic Properties of Nucleic Acids

HIGHLIGHT 6-2 MEDICINE RNA Structure Governing HIV Gene Expression

Double-Helical DNA and RNA Can Be Denatured

Nucleic Acids from Different Species Can Form Hybrids

Nucleotides and Nucleic Acids Undergo Uncatalyzed Chemical Transformations

Base Methylation in DNA Plays an Important Role in Regulating Gene Expression

RNA Molecules Are Often Site-Specifically Modified In Vivo

The Chemical Synthesis of DNA and RNA Has Been Automated

HOW WE KNOW

DNA Is a Double Helix

DNA Helices Have Unique Geometries That Depend on Their Sequence

Ribosomal RNA Sequence Comparisons Provided the First Hints of the Structural Richness of RNA

7

Studying Genes

MOMENT OF DISCOVERY Norman Arnheim, on the discovery of interspersed CA repeats in genomic DNA

7.1 Isolating Genes for Study (Cloning)

Genes Are Cloned by Insertion into Cloning Vectors

Cloning Vectors Allow Amplification of Inserted DNA Segments

DNA Libraries Provide Specialized Catalogs of Genetic Information

7.2 Working with Genes and Their Products

Gene Sequences Can Be Amplified with the Polymerase Chain Reaction

HIGHLIGHT 7-1 TECHNOLOGY A Potent Weapon in Forensic Medicine

The Sanger Method Identifies Nucleotide Sequences in Cloned Genes

Genomic Sequencing Is Aided by New Generations of DNA Sequencing Methods

Cloned Genes Can Be Expressed to Amplify Protein Production

Many Different Systems Are Used to Express Recombinant Proteins

Alteration of Cloned Genes Produces Altered Proteins

Terminal Tags Provide Handles for Affinity Purification

7.3 Understanding the Functions of Genes and Their Products

Protein Fusions and Immunofluorescence Can Localize Proteins in Cells

Proteins Can Be Detected in Cellular Extracts with the Aid of Western Blots

Protein-Protein Interactions Can Help Elucidate Protein Function

DNA Microarrays Reveal Cellular Protein Expression Patterns and Other Information

A Gene’s Function Can Be Elucidated by Examining the Effects of Its Absence

HOW WE KNOW

New Enzymes Take Molecular Biologists from Cloning to Genetically Modified Organisms

A Dreamy Night Ride on a California Byway Gives Rise to the Polymerase Chain Reaction

Coelenterates Show Biologists the Light

8

Genomes, Transcriptomes, and Proteomes

MOMENT OF DISCOVERY Joe DeRisi, on his discovery of the SARS virus

8.1 Genomes and Genomics

Many Genomes Have Been Sequenced in Their Entirety

Annotation Provides a Description of the Genome

Genome Databases Provide Information about Every Type of Organism

HIGHLIGHT 8-1 TECHNOLOGY Sampling Biodiversity with Metagenomics

The Human Genome Contains Many Types of Sequences

Genome Sequencing Informs Us about Our Humanity

Genome Comparisons Help Locate Genes Involved in Disease

8.2 Transcriptomes and Proteomes

Special Cellular Functions Are Revealed in a Cell’s Transcriptome

High-Throughput DNA Sequencing Is Used in Transcriptome Analysis

The Proteins Generated by a Cell Constitute Its Proteome

Electrophoresis and Mass Spectrometry Support Proteomics Research

Computational Approaches Help Elucidate Protein Function

Experimental Approaches Reveal Protein Interaction Networks

8.3 Our Genetic History

All Living Things Have a Common Ancestor

Genome Comparisons Provide Clues to Our Evolutionary Past

HIGHLIGHT 8-2 EVOLUTION Phylogenetics Solves a Crime

The Human Journey Began in Africa

Human Migrations Are Recorded in Haplotypes

HIGHLIGHT 8-3 EVOLUTION Getting to Know the Neanderthals

HOW WE KNOW

Haemophilus influenzae Ushers in the Era of Genome Sequences

9

Topology: Functional Deformations of DNA

MOMENT OF DISCOVERY Carlos Bustamante, on discovering the elasticity of DNA

9.1 Chromosomes: An Overview

Chromosome Function Relies on Specialized Genomic Sequences

Chromosomes Are Longer Than the Cellular or Viral Packages Containing Them

HIGHLIGHT 9-1 MEDICINE The Dark Side of Antibiotics

9.2 DNA Supercoiling

Most Cellular DNA Is Underwound

DNA Underwinding Is Defined by the Topological Linking Number

DNA Compaction Requires a Special Form of Supercoiling

9.3 The Enzymes That Promote DNA Compaction

Topoisomerases Catalyze Changes in the Linking Number of DNA

HIGHLIGHT 9-2 MEDICINE Curing Disease by Inhibiting Topoisomerases

The Two Bacterial Type II Topoisomerases Have Distinct Functions

Eukaryotic Topoisomerases Have Specialized Functions in DNA Metabolism

SMC Proteins Facilitate the Condensation of Chromatin

HOW WE KNOW

The Discovery of Supercoiled DNA Goes through Twists and Turns

The First DNA Topoisomerase Unravels Some Mysteries

DNA Gyrase Passes the Strand Test

10

Nucleosomes, Chromatin, and Chromosome Structure

MOMENT OF DISCOVERY C. David Allis, on establishing that p55 from Tetrahymena is a histone acetylase, as is transcription factor Gcn5

10.1 Nucleosomes: The Basic Units of DNA Condensation

Histone Octamers Organize DNA into Repeating Units

DNA Wraps around a Single Histone Octamer

Histone Tails Mediate Internucleosome Connections That Regulate the Accessibility of DNA

10.2 Higher-Order Chromosome Structure

Histone H1 Binds the Nucleosome

Chromosomes Condense into a Compact Chromatin Filament

Higher-Order Chromosome Structure Involves Loops and Coils

Bacterial DNA, Like Eukaryotic DNA, Is Highly Organized

10.3 Regulation of Chromosome Structure

Nucleosomes Are Intrinsically Dynamic

ATP-Driven Chromatin Remodeling Complexes Can Reposition Nucleosomes

Variant Histone Subunits Alter DNA-Binding Affinity

Nucleosome Assembly Requires Chaperones

Modifications of Histone Tails Alter DNA Accessibility

HIGHLIGHT 10-1 A CLOSER LOOK The Use of a Histone Variant in X Chromosome Inactivation

Proteins with Bromodomains and Chromodomains Bind Modified Histones

Histone Modifications and Remodeling Complexes May Read a Histone Code

Histone Modifying Enzymes Maintain Epigenetic States through Cell Division

HIGHLIGHT 10-2 MEDICINE Defects in Epigenetic Maintenance Proteins Associated with Cancer

HOW WE KNOW

Kornberg Wrapped His Mind around the Histone Octamer

A Transcription Factor Can Acetylate Histones

III

INFORMATION TRANSFER

11

DNA Replication

MOMENT OF DISCOVERY Robert Lehman, on discovering DNA ligase

11.1 DNA Transactions during Replication

DNA Replication Is Semiconservative

Replication Is Initiated at Origins and Proceeds Bidirectionally

Replication Is Semidiscontinuous

11.2 The Chemistry of DNA Polymerases

DNA Polymerases Elongate DNA in the 5′→3′ Direction

Most DNA Polymerases Have DNA Exonuclease Activity

Five E. coli DNA Polymerases Function in DNA Replication and Repair

DNA Polymerase Structure Reveals the Basis for Its Accuracy

Processivity Increases the Efficiency of DNA Polymerase Activity

11.3 Mechanics of the DNA Replication Fork

DNA Polymerase III Is the Replicative Polymerase in E. coli

A DNA Sliding Clamp Increases the Speed and Processivity of the Chromosomal Replicase

Many Different Proteins Advance a Replication Fork

Helicase Activity Is Stimulated by Its Connection to the DNA Polymerase

DNA Loops Repeatedly Grow and Collapse on the Lagging Strand

Okazaki Fragments Require Removal of RNA and Ligase-Mediated Joining of DNA

The Replication Fork Is More Complex in Eukaryotes Than in Bacteria

11.4 Initiation of DNA Replication

Assembly of the Replication Fork Follows an Ordered Sequence of Events

Replication Initiation in E. coli Is Controlled at Multiple Steps

Eukaryotic Origins “Fire” Only Once per Cell Cycle

HIGHLIGHT 11-1 TECHNOLOGY Two-Dimensional Gel Analysis of Replication Origins

11.5 Termination of DNA Replication

E. coli Chromosome Replication Terminates Opposite the Origin

Telomeres and Telomerase Solve the End Replication Problem in Eukaryotes

Telomere Length Is Associated with Immortality and Cancer

Telomeres are Protected and Regulated by Proteins

HIGHLIGHT 11-2 MEDICINE Short Telomeres Portend Aging Diseases

HOW WE KNOW

DNA Polymerase Reads the Sequence of the DNA Template to Copy the DNA

Polymerase Processivity Depends on a Circular Protein That Slides along DNA

Replication Requires an Origin

12

DNA Mutation and Repair

MOMENT OF DISCOVERY Rose Byrne, on her discovery that E. coli could become a radiation-resistant extremophile

12.1 Types of DNA Mutations

A Point Mutation Can Alter One Amino Acid

Small Insertion and Deletion Mutations Change Protein Length

Some Mutations Are Very Large and Form Abnormal Chromosomes

12.2 DNA Alterations That Lead to Mutations

Spontaneous DNA Damage by Water Can Cause Point Mutations

Oxidative Damage and Alkylating Agents Can Create Point Mutations and Strand Breaks

The Ames Test Identifies DNA-Damaging Chemicals

DNA-Damaging Agents Are Used in Cancer Chemotherapy

Solar Radiation Causes Interbase Cross-Links and Strand Breaks

Errant Replication and Recombination Lead to DNA Damage

12.3 Mechanisms of DNA Repair

Mismatch Repair Fixes Misplaced-Nucleotide Replication Errors

Direct Repair Corrects a Damaged Nucleotide Base in One Step

HIGHLIGHT 12-1 MEDICINE Mismatch Repair and Colon Cancer

Base Excision Repairs Subtle Alterations in Nucleotide Bases

Nucleotide Excision Repair Removes Bulky Damaged Bases

HIGHLIGHT 12-2 MEDICINE Nucleotide Excision Repair and Xeroderma Pigmentosum

Recombination Repairs Lesions That Break DNA

Specialized Translesion DNA Polymerases Extend DNA Past a Lesion

HOW WE KNOW

Mismatch Repair in E. coli Requires DNA Methylation

UV Lights Up the Pathway to DNA Damage Repair

Translesion DNA Polymerases Produce DNA Mutations

13

Recombinational DNA Repair and Homologous Recombination

MOMENT OF DISCOVERY Lorraine Symington, on discovering how DNA ends are processed to initiate DNA recombination

13.1 Recombination as a DNA Repair Process

Double-Strand Breaks Are Repaired by Recombination

Collapsed Replication Forks Are Reconstructed by Double-Strand Break Repair

A Stalled Replication Fork Requires Fork Regression

Single-Stranded DNA Regions Are Filled In by Gap Repair

13.2 Enzymatic Machines in Bacterial Recombinational DNA Repair

RecBCD and RecFOR Initiate Recombinational Repair

RecA Protein Is the Bacterial Recombinase

RecA Protein Is Subject to Regulation

Multiple Enzymes Process DNA Intermediates Created by RecA

HIGHLIGHT 13-1 EVOLUTION A Tough Organism in a Tough Environment: Deinococcus radiodurans

Repair of the Replication Fork in Bacteria Can Lead to Dimeric Chromosomes

13.3 Homologous Recombination in Eukaryotes

HIGHLIGHT 13-2 MEDICINE Why Proper Chromosomal Segregation Matters

Meiotic Recombination Is Initiated at Double-Strand Breaks

Meiotic Recombination Is Completed by a Classic DSBR Pathway

Meiotic Recombination Contributes to Genetic Diversity

Recombination during Mitosis Is Also Initiated at Double-Strand Breaks

Programmed Gene Conversion Events Can Affect Gene Function and Regulation

Some Introns Move via Homologous Recombination

13.4 Nonhomologous End Joining

Nonhomologous End Joining Repairs Double-Strand Breaks

Nonhomologous End Joining Is Promoted by a Set of Conserved Enzymes

Recombination Systems Are Being Harnessed for Genome Editing

HOW WE KNOW

A Motivated Graduate Student Inspires the Discovery of Recombination Genes in Bacteria

A Biochemical Masterpiece Catches a Recombination Protein in the Act

14

Site-Specific Recombination and Transposition

MOMENT OF DISCOVERY Wei Yang, on researching the structure and molecular mechanisms of γδ resolvase

14.1 Mechanisms of Site-Specific Recombination

Precise DNA Rearrangements Are Promoted by Site-Specific Recombinases

Site-Specific Recombination Complements Replication

Site-Specific Recombination Can Be a Stage in a Viral Infection Cycle

Site-Specific Recombination Systems Are Used in Biotechnology

Gene Expression Can Be Regulated by Site-Specific Recombination

HIGHLIGHT 14-1 TECHNOLOGY Using Site-Specific Recombination to Trace Neurons

14.2 Mechanisms of Transposition

Transposition Takes Place by Three Major Pathways

Bacteria Have Three Common Classes of Transposons

Retrotransposons Are Especially Common in Eukaryotes

HIGHLIGHT 14-2 EVOLUTION Awakening Sleeping Beauty

Retrotransposons and Retroviruses Are Closely Related

A Retrovirus Causes AIDS

HIGHLIGHT 14-3 MEDICINE Fighting AIDS with HIV Reverse Transcriptase Inhibitors

14.3 The Evolutionary Interplay of Transposons and Their Hosts

Viruses, Transposons, and Introns Have an Interwoven Evolutionary History

A Hybrid Recombination Process Assembles Immunoglobulin Genes

HOW WE KNOW

Bacteriophage λ Provided the First Example of Site-Specific Recombination

If You Leave Out the Polyvinyl Alcohol, Transposition Gets Stuck

15

Transcription: DNA-Dependent Synthesis of RNA

MOMENT OF DISCOVERY Robert Tjian, on discovering the first specific eukaryotic transcription factor

15.1 RNA Polymerases and Transcription Basics

RNA Polymerases Differ in Details but Share Many Features

HIGHLIGHT 15-1 A CLOSER LOOK The ABCs of RNA: Complexity of the Transcriptome

Transcription Initiation, Elongation, and Termination Occur in Discrete Steps

DNA-Dependent RNA Polymerases Can Be Specifically Inhibited

Transcriptional Regulation Is a Central Mechanism in the Control of Gene Expression

15.2 Transcription in Bacteria

Promoter Sequences Alter the Strength and Frequency of Transcription

Sigma Factors Specify Polymerase Binding to Particular Promoters

Structural Changes Lead to Formation of the Transcription-Competent Open Complex

Initiation Is Primer-Independent and Produces Short, Abortive Transcripts

Transcription Elongation Is Continuous until Termination

Specific Sequences in the Template Strand Stop Transcription

15.3 Transcription in Eukaryotes

Eukaryotic Polymerases Recognize Characteristic Promoters

HIGHLIGHT 15-2 MEDICINE Using Transcription Factors to Reprogram Cells

Pol II Transcription Parallels Bacterial RNA Transcription

Transcription Factors Play Specific Roles in the Transcription Process

Transcription Initiation In Vivo Requires the Mediator Complex

Termination Mechanisms Vary among RNA Polymerases

Transcription Is Coupled to DNA Repair, RNA Processing, and mRNA Transport

HOW WE KNOW

RNA Polymerase Is Recruited to Promoter Sequences

RNA Polymerases Are Both Fast and Slow

16

RNA Processing

MOMENT OF DISCOVERY Melissa Jurica, on determining the first electron microscopic structures of spliceosomes

16.1 Messenger RNA Capping and Polyadenylation

Eukaryotic mRNAs Are Capped at the 5′ End

Eukaryotic mRNAs Have a Distinctive 3′-End Structure

mRNA Capping, Polyadenylation, and Splicing Are Coordinately Regulated during Transcription

HIGHLIGHT 16-1 EVOLUTION Eukaryotic mRNAs with Unusual 3′ Tails

16.2 Pre-mRNA Splicing and Editing

Eukaryotic mRNAs Are Synthesized as Precursors Containing Introns

Alternative RNA Splicing Can Generate Multiple Products from a Gene

The Spliceosome Catalyzes Most Pre-mRNA Splicing

Some Introns Can Self-Splice without Protein or Spliceosome Assistance

Exons from Different RNA Molecules Can Be Fused by Trans-Splicing

RNA Editing Can Involve the Insertion or Deletion of Bases

HIGHLIGHT 16-2 EVOLUTION The Origin of Introns

RNA Editing by Substitution Involves Deamination of A or C Residues

16.3 RNA Transport and Degradation

Different Kinds of RNA Use Different Nuclear Export Pathways

mRNA Export from the Nucleus Is Coupled to Pre-mRNA Splicing

Some mRNAs Are Localized to Specific Regions of the Cytoplasm

Cellular mRNAs Are Degraded at Different Rates

Processing Bodies Are the Sites of mRNA Storage and Degradation in Eukaryotic Cells

16.4 Processing of Non-Protein-Coding RNAs

Maturation of tRNAs Involves Site-Specific Cleavage and Chemical Modification

Maturation of rRNA Involves Site-Specific Cleavage and Chemical Modification

Small Regulatory RNAs Are Derived from Larger Precursor Transcripts

16.5 RNA Catalysis and the RNA World Hypothesis

Ribozymes Catalyze Similar Kinds of Reactions But Have Diverse Functions

HIGHLIGHT 16-3 EVOLUTION A Viral Ribozyme Derived from the Human Genome?

Could RNA Have Formed the Basis for Early Life on Earth?

HOW WE KNOW

Studying Autoimmunity Led to the Discovery of snRNPs

RNA Molecules Are Fine-Tuned for Stability or Function

Ribozyme Form Explains Function

17

The Genetic Code

MOMENT OF DISCOVERY Steve Benner, on discovering that borate minerals stabilize ribose

17.1 Deciphering the Genetic Code: tRNA as Adaptor

All tRNAs Have a Similar Structure

The Genetic Code Is Degenerate

Wobble Enables One tRNA to Recognize Two or More Codons

Specific Codons Start and Stop Translation

The Genetic Code Resists Single-Base Substitution Mutations

Some Mutations Are Suppressed by Special tRNAs

17.2 The Rules of the Code

The Genetic Code Is Nonoverlapping

There Are No Gaps in the Genetic Code

The Genetic Code Is Read in Triplets

Protein Synthesis Is Linear

17.3 Cracking the Code

Random Synthetic RNA Polymers Direct Protein Synthesis in Cell Extracts

RNA Polymers of Defined Sequence Complete the Code

The Genetic Code Is Validated in Living Cells

17.4 Exceptions Proving the Rules

Evolution of the Translation Machinery Is a Mystery

Mitochondrial tRNAs Deviate from the Universal Genetic Code

HIGHLIGHT 17-1 EVOLUTION The Translation Machinery

Initiation and Termination Rules Have Exceptions

HOW WE KNOW

Transfer RNA Connects mRNA and Protein

Proteins Are Synthesized from the N-Terminus to the C-Terminus

The Genetic Code In Vivo Matches the Genetic Code In Vitro

18

Protein Synthesis

MOMENT OF DISCOVERY Harry Noller, on discovering the functional importance of ribosomal RNA

18.1 The Ribosome

The Ribosome Is an RNA-Protein Complex Composed of Two Subunits

Ribosomal Subunits Associate and Dissociate in Each Cycle of Translation

The Ribosome Is a Ribozyme

The Ribosome Structure Facilitates Peptide Bond Formation

HIGHLIGHT 18-1 EVOLUTION Mitochondrial Ribosomes: A Window into Ribosome Evolution?

18.2 Activation of Amino Acids for Protein Synthesis

Amino Acids Are Activated and Linked to Specific tRNAs

Aminoacyl-tRNA Synthetases Attach the Correct Amino Acids to Their tRNAs

The Structure of tRNA Allows Accurate Recognition by tRNA Synthetases

Proofreading Ensures the Fidelity of Aminoacyl-tRNA Synthetases

18.3 Initiation of Protein Synthesis

HIGHLIGHT 18-2 TECHNOLOGY Genetic Incorporation of Unnatural Amino Acids into Proteins

Base Pairing Recruits the Small Ribosomal Subunit to Bacterial mRNAs

Eukaryotic mRNAs Recruit the Small Ribosomal Subunit Indirectly

A Specific Amino Acid Initiates Protein Synthesis

Initiation in Bacterial Cells Requires Three Initiation Factors

Initiation in Eukaryotic Cells Requires Additional Initiation Factors

Some mRNAs Use 5’ End–Independent Mechanisms of Initiation

18.4 Elongation and Termination of the Polypeptide Chain

Peptide Bonds Are Formed in the Translation Elongation Stage

Substrate Positioning and the Incoming tRNA Contribute to Peptide Bond Formation

EF-G Drives Translocation by Displacing the A-Site tRNA

GTP Binding and Hydrolysis Regulate Successive Elongation Cycles

An mRNA Stop Codon Signals Completion of a Polypeptide Chain

Ribosome Recycling Factor Prepares Ribosomes for New Rounds of Translation

Fast and Accurate Protein Synthesis Requires Energy

Antibiotics and Toxins Frequently Target Protein Synthesis

HIGHLIGHT 18-3 MEDICINE Toxins That Target the Ribosome

18.5 Translation-Coupled Removal of Defective mRNA

Ribosomes Stalled on Truncated mRNAs Are Rescued by tmRNA

Eukaryotes Have Other Mechanisms to Detect Defective mRNAs

18.6 Protein Folding, Covalent Modification, and Targeting

Protein Folding Sometimes Requires the Assistance of Chaperones

Covalent Modifications Are Common in Newly Synthesized Proteins

Proteins Are Targeted to Correct Locations during or after Synthesis

Some Chemical Modifications of Eukaryotic Proteins Take Place in the Endoplasmic Reticulum

Glycosylation Plays a Key Role in Eukaryotic Protein Targeting

Signal Sequences for Nuclear Transport Are Not Removed

Bacteria Also Use Signal Sequences for Protein Targeting

HOW WE KNOW

The Ribosome Is a Ribozyme

Ribosomes Check the Accuracy of Codon-Anticodon Pairing, but Not the Identity of the Amino Acid

IV

REGULATION

19

Regulating the Flow of Information

MOMENT OF DISCOVERY Lin He, on discovering that microRNA overexpression accelerates tumor development

19.1 Regulation of Transcription Initiation

Activators and Repressors Control RNA Polymerase Function at a Promoter

Transcription Factors Can Function by DNA Looping

Regulators Often Work Together for Signal Integration

Gene Expression Is Regulated through Feedback Loops

Related Sets of Genes Are Often Regulated Together

Eukaryotic Promoters Use More Regulators Than Bacterial Promoters

Multiple Regulators Provide Combinatorial Control

Regulation by Nucleosomes Is Specific to Eukaryotes

19.2 The Structural Basis of Transcriptional Regulation

Transcription Factors Interact with DNA and Proteins through Structural Motifs

Transcription Activators Have Separate DNA-Binding and Regulatory Domains

19.3 Posttranscriptional Regulation of Gene Expression

Some Regulatory Mechanisms Act on the Nascent RNA Transcript

Small RNAs Can Affect mRNA Stability

Some Genes Are Regulated at the Level of Translation

Some Covalent Modifications Regulate Protein Function

Gene Expression Can Be Regulated by Intracellular Localization

HIGHLIGHT 19-1 MEDICINE Insulin Regulation: Control by Phosphorylation

Protein Degradation by Ubiquitination Modulates Gene Expression

HOW WE KNOW

Plasmids Have the Answer to Enhancer Action

20

The Regulation of Gene Expression in Bacteria

MOMENT OF DISCOVERY Bonnie Bassler, on her discovery of interspecies quorum sensing

20.1 Transcriptional Regulation

The lac Operon Is Subject to Negative Regulation

The lac Operon Also Undergoes Positive Regulation

HIGHLIGHT 20-1 TECHNOLOGY Classical Techniques in the Analysis of Gene Regulation

CRP Functions with Activators or Repressors to Control Gene Transcription

Transcription Attenuation Often Controls Amino Acid Biosynthesis

The SOS Response Leads to Coordinated Transcription of Many Genes

20.2 Beyond Transcription: Control of Other Steps in the Gene Expression Pathway

RNA Sequences or Structures Can Control Gene Expression Levels

Translation of Ribosomal Proteins Is Coordinated with rRNA Synthesis

HIGHLIGHT 20-2 A CLOSER LOOK T-Box Riboswitches

20.3 Control of Gene Expression in Bacteriophages

Phage Propagation Can Take One of Two Forms

Differential Activation of Promoters Regulates λ Phage Infection

The λ Repressor Functions as Both an Activator and a Repressor

More Regulation Levels Are Invoked during the λ Phage Life Cycle

HOW WE KNOW

TRAPped RNA Inhibits Expression of Tryptophan Biosynthetic Genes in Bacillus subtilis

Autoinducer Analysis Reveals Possibilities for Treating Cholera

21

The Transcriptional Regulation of Gene Expression in Eukaryotes

MOMENT OF DISCOVERY Tracy Johnson, on discovering that pre-mRNA splicing requires specific histone acetylation

21.1 Basic Mechanisms of Eukaryotic Transcriptional Activation

Eukaryotic Transcription Is Regulated by Chromatin Structure

Positive Regulation of Eukaryotic Promoters Involves Multiple Protein Activators

HIGHLIGHT 21-1 A CLOSER LOOK The Intertwining of Transcription and mRNA Splicing

Transcription Activators and Coactivators Help Assemble General Transcription Factors

21.2 Combinatorial Control of Gene Expression

Combinatorial Control of the Yeast GAL Genes Involves Positive and Negative Regulation

HIGHLIGHT 21-2 TECHNOLOGY Discovering and Analyzing DNA-Binding Proteins

Combinatorial Control of Transcription Causes Mating-Type Switches in Yeast

Combinatorial Mixtures of Heterodimers Regulate Transcription

Differentiation Requires Extensive Use of Combinatorial Control

21.3 Transcriptional Regulation Mechanisms Unique to Eukaryotes

Insulators Separate Adjacent Genes in a Chromosome

Some Activators Assemble into Enhanceosomes

Gene Silencing Can Inactivate Large Regions of Chromosomes

Imprinting Allows Selective Gene Expression from One Allele Only

HIGHLIGHT 21-3 A CLOSER LOOK Gene Silencing by Small RNAs

Dosage Compensation Balances Gene Expression from Sex Chromosomes

Steroid Hormones Bind Nuclear Receptors That Regulate Gene Expression

Nonsteroid Hormones Control Gene Expression by Triggering Protein Phosphorylation

HOW WE KNOW

Transcription Factors Bind Thousands of Sites in the Fruit Fly Genome

Muscle Tissue Differentiation Reveals Surprising Plasticity in the Basal Transcription Machinery

22

The Posttranscriptional Regulation of Gene Expression in Eukaryotes

MOMENT OF DISCOVERY Judith Kimble, on the discovery that noncoding regions of mRNA regulate cell fate

22.1 Posttranscriptional Control inside the Nucleus

Alternative Splicing Controls Sex Determination in Fruit Flies

Multiple mRNA Cleavage Sites Allow the Production of Multiple Proteins

Nuclear Transport Regulates Which mRNAs Are Selected for Translation

22.2 Translational Control in the Cytoplasm

Initiation Can Be Suppressed by Phosphorylation of eIF

The 3′UTR of Some mRNAs Controls Translational Efficiency

Upstream Open Reading Frames Control the Translation of GCN4 mRNA

mRNA Degradation Rates Can Control Translational Efficiency

22.3 The Large-Scale Regulation of Groups of Genes

Some Sets of Genes Are Regulated by Pre-mRNA Splicing in the Nucleus

5′UTRs and 3′UTRs Coordinate the Translation of Multiple mRNAs

HIGHLIGHT 22-1 EVOLUTION Regulation of Splicing in Response to Stress

Conserved AU-Rich Elements in 3′UTRs Control Global mRNA Stability for Some Genes

22.4 RNA Interference

Eukaryotic MicroRNAs Target mRNAs for Gene Silencing

Short Interfering RNAs Target mRNAs for Degradation

RNAi Pathways Regulate Viral Gene Expression

RNAi Provides a Useful Tool for Molecular Biologists

HIGHLIGHT 22-2 MEDICINE Viral Takeover Using a Cell Type–Specific miRNA

RNAs Regulate a Wide Range of Cellular Processes

22.5 Putting It All Together: Gene Regulation in Development

Development Depends on Asymmetric Cell Divisions and Cell-Cell Signaling

Early Development Is Mediated by Maternal Genes

Segmentation Genes Specify the Development of Body Segments and Tissues

Homeotic Genes Control the Development of Organs and Appendages

Stem Cells Have Developmental Potential That Can Be Controlled

22.6 Finale: Molecular Biology, Developmental Biology, and Evolution

The Interface of Evolutionary and Developmental Biology Defines a New Field

Small Genetic Differences Can Produce Dramatic Phenotypic Changes

HOW WE KNOW

A Natural Collaboration Reveals a Binding Protein for a 3′UTR

Little RNAs Play a Big Role in Controlling Gene Expression

Everything Old Is New Again: Beauty at the Turn of a Developmental Switch

Model Organisms Appendix

A Few Organisms Are Models for Understanding Common Life Processes

Three Approaches Are Used to Study Human Disease

Bacterium, Escherichia coli

Early Studies of E. coli as a Model Organism

Life Cycle

Genetic Techniques

E. coli as a Model Organism Today

Budding Yeast, Saccharomyces cerevisiae

Early Studies of Yeast as a Model Organism

Life Cycle

Genetic Techniques

Yeast as a Model Organism Today

Bread Mold, Neurospora crassa

Early Studies of Neurospora as a Model Organism

Life Cycle

Genetic Techniques

Neurospora as a Model Organism Today

Nematode, Caenorhabditis elegans

Early Studies of C. elegans as a Model Organism

Life Cycle

Genetic Techniques

C. elegans as a Model Organism Today

Mustard Weed, Arabidopsis thaliana

Early Studies of Arabidopsis as a Model Organism

Life Cycle

Genetic Techniques

Arabidopsis as a Model Organism Today

Fruit Fly, Drosophila melanogaster

Early Studies of Drosophila as a Model Organism

Life Cycle

Genetic Techniques

Drosophila as a Model Organism Today

House Mouse, Mus musculus

Early Studies of the Mouse as a Model Organism

Life Cycle

Genetic Techniques

The Mouse as a Model Organism Today

Glossary

Solutions to Problems

Index