Molecular biology : genes to proteins / 3rd ed.

Table Of Contents:
Preface xix

Introduction to Molecular Biology 1(26)

Intellectual Foundation 2(2)

Two studies performed in the 1860s provided the intellectual underpinning for molecular biology. 2(2)

Genotypes and Phenotypes 4(1)

Each gene is responsible for the synthesis of a single polypeptide. 4(1)

Nucleic Acids 5(1)

Nucleic acids are linear chains of nucleotides. 5(1)

DNA Structure and Function 6(21)

Transformation experiments led to the discovery that DNA is the hereditary material. 12(3)

Chemical experiments also supported the hypothesis that DNA is the hereditary material. 15(1)

The blender experiment demonstrated that DNA is the genetic material in bacterial viruses. 16(1)

RNA serves as the hereditary material in some viruses. 17(2)

Rosalind Franklin and Maurice Wilkins obtained x-ray diffraction patterns of extended DNA fibers. 19(1)

James Watson and Francis Crick proposed that DNA is a double-stranded helix. 19(4)

The central dogma provides the theoretical framework for molecular biology. 23(1)

Recombinant DNA technology allows us to study complex biological systems. 23(2)

A great deal of molecular biology information is available on the Internet. 25(1)

Suggested Reading 26(1)

Classic Papers 26(1)

SECTION 1 Protein Structure and Function 27(72)

Protein Structure 28(43)

The α-Amino Acids 29(4)

α-Amino acids have an amino group and a carboxyl group attached to a central carbon atom. 29(3)

Amino acids are represented by three-letter and one-letter abbreviations. 32(1)

The Peptide Bond 33(1)

α-Amino acids are linked by peptide bonds. 33(1)

Protein Purification 34(7)

Protein mixtures can be fractionated by chromatography. 34(6)

Proteins and other charged biological polymers migrate in an electric field. 40(1)

Primary Structure of Proteins 41(5)

Amino acid sequences can be determined by using the Edman degradation procedure and the overlap method. 41(2)

Polypeptide sequences can be obtained from nucleic acid sequences. 43(2)

The BLAST program compares a new polypeptide sequence with all sequences stored in a data bank. 45(1)

Proteins with just one polypeptide chain have primary, secondary, and tertiary structures while those with two or more chains also have quaternary structures. 45(1)

Weak Non-Covalent Bonds 46(5)

The polypeptide folding pattern is determined by weak non-covalent interactions. 46(5)

Secondary Structures 51(5)

The α-helix is a compact structure that is stabilized by hydrogen bonds. 51(2)

The β-conformation is also stabilized by hydrogen bonds. 53(1)

Loops and turns connect different peptide segments, allowing polypeptide chains to fold back on themselves. 54(1)

Certain combinations of secondary structures, called supersecondary structures or folding motifs, appear in many different proteins. 54(1)

We cannot yet predict secondary structures with absolute certainty. 55(1)

Tertiary Structure 56(7)

X-ray crystallography and nuclear magnetic resonance studies have revealed the three-dimensional structures of many different proteins. 56(3)

The primary structure of a polypeptide determines its tertiary structure. 59(2)

Molecular chaperones help proteins to fold inside the cell. 61(2)

Proteins and Biological Membranes 63(8)

Proteins interact with lipids in biological membranes. 63(2)

The fluid mosaic model has been proposed to explain the structure of biological membranes. 65(3)

Suggested Reading 68(3)

Protein Function 71(28)

Myoglobin, Hemoglobin, and the Quaternary Structure Concept 72(6)

Differences in myoglobin and hemoglobin function are explained by differences in myoglobin and hemoglobin structure. 72(4)

Normal adult hemoglobin (HbA) differs from sickle cell hemoglobin (HbS) by only one amino acid. 76(2)

Immunoglobulin G and the Domain Concept 78(2)

Large polypeptides fold into globular units called domains. 78(2)

Enzymes 80(8)

Enzymes are proteins that catalyze chemical reactions. 80(1)

Different methods can be used to detect enzyme activity. 81(1)

Enzymes lower the energy of activation but do not affect the equilibrium position. 82(1)

All enzyme reactions proceed through an enzyme-substrate complex. 83(2)

Molecular details for enzyme-substrate complexes have been worked out for many enzymes. 85(1)

Regulatory enzymes control committed steps in biochemical pathways. 85(2)

Regulatory enzymes exhibit sigmoidal kinetics and are stimulated or inhibited by allosteric effectors. 87(1)

Enzyme activity can be altered by covalent modification. 88(1)

G Protein Signal System 88(11)

G protein signal systems transmit external signals into the cell. 88(8)

Suggested Reading 96(3)

SECTION 2 Nucleic Acids and Nucleoproteins 99(112)

Deoxyribonucleic Acid Structure 100(30)

Size Variation 101(1)

DNA molecules vary in size and base composition. 101(1)

Fragility 102(1)

DNA molecules are fragile. 102(1)

DNA Conformation 102(5)

A linear double-stranded DNA molecule can exist in one of three conformations, A-DNA, B-DNA, or Z-DNA. 102(4)

Base sequences can influence DNA structure. 106(1)

DNA Denaturation 107(6)

DNA can be denatured. 107(2)

Hydrogen bonds stabilize double-stranded DNA. 109(1)

Base stacking also stabilizes double-stranded DNA. 110(1)

Base stacking is a cooperative interaction. 110(1)

Ionic strength influences DNA structure. 111(1)

The DNA molecule is in a dynamic state. 111(1)

Distant short patches of complementary sequences can base pair in single-stranded DNA. 112(1)

Alkali denatures DNA without breaking phosphodiester bonds. 112(1)

Renaturation 113(1)

Complementary single-strands can anneal to form double-stranded DNA. 113(1)

Helicases 114(2)

Helicases are motor proteins that use the energy of nucleoside triphosphates to unwind DNA. 114(2)

Single-Strand DNA Binding Proteins 116(2)

Single-strand DNA binding proteins (SSB) stabilize single-stranded DNA. 116(2)

Topoisomers and Topoisomerases 118(12)

Most bacterial DNA molecules and many viral DNA molecules are circular. 118(1)

Circular DNA molecules often have superhelical structures. 118(1)

Supercoiled DNA results from under- or overwinding circular DNA. 119(3)

Superhelices can have single-stranded regions. 122(1)

Topoisomerases catalyze the conversion of one topoisomer into another. 122(1)

Enzymes belonging to the topoisomerase I family can be divided into two subfamilies. 123(1)

Type II topoisomerases require ATP to convert one topoisomer into another. 124(3)

Suggested Reading 127(3)

Nucleic Acid Technology 130(44)

Nucleic Acid Isolation 131(2)

The method used for DNA isolation must be tailored to the organism from which the DNA is to be isolated. 131(1)

Great care must be taken to protect RNA from degradation during its isolation. 132(1)

Different physical techniques are used to study macromolecules. 133(1)

Electron Microscopy 133(2)

Electron microscopy allows us to see macromolecules. 133(2)

Centrifugal Techniques 135(4)

Velocity sedimentation can separate macromolecules and provide information about their size and shape. 135(3)

Equilibrium density gradient centrifugation separates particles according to their density. 138(1)

Gel Electrophoresis 139(5)

Gel electrophoresis separates charged macromolecules by their rate of migration in an electric field. 139(3)

Gel electrophoresis can be used to separate proteins and determine the molecular mass of a polypeptide. 142(1)

Pulsed-field gel electrophoresis (PFGE) can separate very large DNA molecules. 143(1)

Nucleases and Restriction Maps 144(8)

Nucleases are useful tools in DNA investigations. 144(1)

Restriction endonucleases that cleave within specific nucleotide sequences are very useful tools for characterizing DNA. 145(4)

Restriction endonucleases can be used to construct a restriction map of a DNA molecule. 149(3)

Recombinant DNA Technology 152(22)

DNA fragments can be inserted into plasmid DNA vectors. 152(2)

Southern blotting is used to detect specific DNA fragments. 154(2)

Northern and Western blotting are used to detect specific RNA and polypeptide molecules, respectively. 156(1)

DNA polymerase I, a multifunctional enzyme with polymerase, 3'→5' exonuclease, and 5'→3' exonuclease activities, can be used to synthesize labeled DNA. 156(4)

DNA polymerase I can synthesize DNA at a nick. 160(2)

The polymerase chain reaction is used to amplify DNA. 162(1)

Site-directed mutagenesis can be used to introduce a specific base change within a gene. 163(2)

Frederick Sanger devised the dideoxynucleotide method for sequencing DNA. 165(2)

Reverse transcriptase can use an RNA molecule as a template to synthesize DNA. 167(2)

DNA chips are used to follow mRNA synthesis, search for a specific DNA sequence, or to find a single nucleotide change in a DNA sequence. 169(2)

Suggested Reading 171(3)

Chromosome Structure 174(37)

Bacterial Chromatin 176(3)

Specific proteins help to condense bacterial DNA. 176(1)

SMC proteins make an important contribution to the compaction of the bacterial chromosome. 177(2)

Mitosis and Meiosis 179(8)

In higher animals, germ cells have a haploid number of chromosomes and somatic cells have a diploid number. 179(1)

The animal cell life cycle alternates between interphase and mitosis. 180(1)

Mitosis allows cells to maintain the chromosome number. 181(2)

Meiosis reduces the chromosome number in half. 183(4)

Karyotype 187(3)

Chromosome sites are specified according to nomenclature conventions. 187(1)

A karyotype shows an individual cell's metaphase chromosomes arranged in pairs and sorted by size. 187(1)

A great deal of information can be obtained by examining karyotype preparations. 188(1)

Fluorescent in situ hybridization (FISH) provides a great deal of information about chromosomes. 189(1)

The Nucleosome 190(5)

Five major histone classes interact with DNA in eukaryotic chromatin. 190(1)

Chromatin is made of bead-like structures called nucleosomes. 191(2)

X-ray crystallography provides high-resolution images of nucleosome core particles. 193(2)

Nucleosome assembly is tightly coupled to DNA replication. 195(1)

Higher Levels of Eukaryotic Chromatin Organization 195(16)

Nucleosomes fold into a three-dimensional structure in which the linker DNA has a zigzag pattern. 195(6)

The centromere is the site of microtubule attachment. 201(1)

The telomere, which is present at either end of a chromosome, is needed for stability. 202(4)

Suggested Reading 206(5)

SECTION 3 Genetics and Virology 211(106)

Genetic Analysis in Molecular Biology 212(51)

Introduction to Genetic Recombination 213(2)

Genetic recombination involves an exchange of DNA segments between DNA molecules or chromosomes. 213(1)

Recombination frequencies are used to obtain a genetic map. 214(1)

Bacterial Genetics 215(27)

Bacteria, which are often selected as model systems for genetic analyses, have complex structures. 215(1)

Bacteria can be cultured in liquid or solid media. 216(4)

Specific notations, conventions, and terminology are used in bacterial genetics. 220(1)

Cells with altered genes are called mutants. 221(1)

Some mutants display the mutant phenotype under all conditions, while others display it only under certain conditions. 221(1)

Certain physical and chemical agents are mutagens. 222(1)

Mutants can be classified on the basis of the changes in the DNA. 222(1)

A mutant organism may regain its original phenotype. 222(1)

Mutants have many uses in molecular biology. 223(3)

A genetic test known as complementation can be used to determine the number of genes responsible for a phenotype. 226(4)

E. coli cells can exchange genetic information by conjugation. 230(2)

The F plasmid can integrate into a bacterial chromosome and carry it into a recipient cell. 232(3)

Bacterial mating experiments can be used to produce an E. coli genetic map. 235(1)

F' plasmids contain part of the bacterial chromosome. 236(3)

A wide variety of plasmids exists in nature. 239(3)

Budding Yeast (Saccharomyces cerevisiae) 242(7)

Yeasts are unicellular eukaryotes. 242(2)

Specific notations, conventions, and terminology are used in yeast genetics. 244(1)

The yeast cells exist in haploid and diploid stages. 244(3)

The yeast mating type is determined by an allele present in the mating type (Mat) locus. 247(1)

Yeast mating factors act as signals to initiate the mating process. 247(2)

Restriction and Amplified Fragment Length Polymorphisms 249(4)

Recombinant DNA techniques have facilitated genetic analysis in humans and other organisms. 249(4)

Somatic Cell Genetics 253(10)

Somatic cell genetics can be used to map genes in higher organisms. 253(1)

Animal cells can be studied in culture. 254(2)

Two different animal cells can fuse to form a heterokaryon. 256(2)

Hybrid cells can be used to make monoclonal antibodies. 258(3)

Suggested Reading 261(2)

Viruses in Molecular Biology 263(54)

Introduction to Viruses 264(2)

Viruses are obligate parasites that can only replicate in a host cell. 264(2)

Introduction to the Bacteriophages 266(5)

Bacteriophages were of interest because they seemed to have the potential to serve as therapeutic agents to treat bacterial diseases. 266(1)

Investigators belonging to the ``Phage Group'' were the first to use viruses as model systems to study fundamental questions about gene structure and function. 266(1)

Bacteriophages come in different sizes and shapes. 267(1)

Bacteriophages have lytic, lysogenic, and chronic life cycles. 268(1)

Bacteriophages form plaques on a bacterial lawn. 268(1)

A specific bacteriophage usually replicates in a very limited number of different kinds of host cells. 269(2)

Virulent Bacteriophages 271(19)

E. coli phage T4 DNA is terminally redundant and circularly permuted. 271(10)

E. coli phage T7 DNA is terminally redundant but not circularly permuted. 281(3)

E. coli phage φX174 contains a single-stranded circular DNA molecule. 284(4)

Some phages have single-stranded RNA as their genetic material. 288(2)

Temperate Phages 290(8)

E. coli phage λ DNA can replicate through a lytic or lysogenic life cycle. 290(7)

E. coli phage P1 can act as generalized transducing particles. 297(1)

Chronic Phages 298(5)

After infection, a chronic phage programs the host cell for continued virion particle release without killing the cell. 298(5)

Animal Viruses 303(14)

Polyomaviruses contain circular double-stranded DNA. 303(2)

Adenonviruses have linear blunt-ended, double-stranded DNA with an inverted repeat at each end. 305(3)

Retroviruses use reverse transcriptase to make a DNA copy of their RNA genome. 308(5)

Suggested Reading 313(4)

SECTION 4 DNA Metabolism 317(236)

DNA Replication 318(61)

General Features of DNA Replication 319(10)

DNA replication is semiconservative. 319(3)

Bacterial and eukaryotic DNA replication is bidirectional. 322(3)

DNA replication is semidiscontinuous. 325(4)

Bacterial DNA Replication Machinery 329(1)

The bacterial replication machinery has been isolated and examined in vitro. 329(1)

Mutant studies provide important information about the enzymes involved in DNA replication. 330(1)

Initiation Stage in Bacteria 330(7)

The replicon model proposes that a site-specific DNA-binding protein binds to a DNA sequence called a replicator. 330(2)

The E. coli initiator protein, DnaA, has four functional domains. 332(1)

E. coli chromosomal replication begins at oriC. 333(2)

Several proteins participate in the initiation of DNA replication. 335(2)

Elongation Stage in Bacteria 337(13)

Several enzymes act together at the replication fork. 337(2)

DNA polymerase III holoenzyme, a complex protein made of three distinct subassemblies, is the bacterial replication machine. 339(2)

The core polymerase subassembly has one subunit with 5'→3' polymerase activity and another with 3'→5' exonuclease activity. 341(2)

The sliding clamp subassembly forms a ring around DNA, tethering the remainder of the polymerase holoenzyme to the DNA. 343(1)

The clamp loader subassembly is a matchmaker that places the sliding clamp around DNA. 344(3)

The clamp loader in DNA polymerase III holoenzyme has two τ subunits in place of two γ subunits. 347(1)

DNA polymerase III holoenzyme catalyzes leading and lagging strand synthesis. 348(2)

Termination Stage in Bacteria 350(1)

Replication terminates when the two growing forks meet in the terminus region, which is located 180° around the circular chromosome from the origin. 350(1)

Eukaryotic DNA Replication Machinery 351(2)

The eukaryotic and prokaryotic DNA replication processes have many features in common. 351(2)

Initiation Stage in Eukaryotes 353(10)

The SV40 T antigen binds to the origin and unwinds DNA. 353(2)

SV40 T antigen helps to recruit DNA polymerase α-primase (Pol α) to the proto-replication bubble. 355(1)

Eukaryotic chromosomes have many replicator sites. 356(1)

Autonomously replicating sequences (ARS) determine the site of DNA chain initiation in yeast. 357(3)

Two-dimensional gel electrophoresis can locate origins of replication. 360(2)

Protein factors are required for DNA initiation in eukaryotes 362(1)

Elongation Stage in Eukaryotes 363(3)

SV40 DNA is replicated by cellular enzymes and only one viral protein, large tumor (T) antigen. 363(3)

Telomeres and Telomerase 366(7)

DNA replication of eukaryotic DNA shortens the leading strand. 366(3)

Telomerase extends the ends of the newly replicated linear duplex. 369(1)

Telomerase RNA functions as a template for DNA synthesis. 370(1)

A telomerase polypeptide is a reverse transcriptase. 371(1)

Telomerase may play a role in aging and cancer. 372(1)

DNA Replication in the Archaea 373(1)

The archaea replication machinery is similar to that in eukaryotes. 373(1)

Initiation Stage in the Archaea 374(1)

Orcl/Cdc6 recruits MCM to the archaeal origin of replication. 374(1)

Elongation Stage in the Archaea 374(5)

The basic steps in archaeal elongation are very similar to those in bacteria and eukaryotes. 374(1)

Suggested Reading 375(4)

DNA Damage and Repair 379(62)

Radiation Damage 381(3)

Ultraviolet light causes cyclobutane pyrimidine dimer (CPD) formation and (6-4) photoproduct formation. 381(2)

X-rays and gamma rays cause many different types of DNA damage. 383(1)

DNA Instability in Water 384(5)

DNA is damaged by hydrolytic cleavage reactions. 384(5)

Oxidative Damage 389(1)

Reactive oxygen species damage DNA. 389(1)

Alkylation Damage by Monoadduct Formation 390(4)

Alkylating agents damage DNA by transferring alkyl groups to centers of negative charge. 390(2)

Many environmental agents must be modified by cell metabolism before they can alkylate DNA. 392(2)

Chemical Cross-Linking Agents 394(3)

Chemical cross-linking agents block DNA strand separation. 394(1)

Psoralen and related compounds can form monoadducts or cross-links. 394(1)

Cisplatin combines with DNA to form intra-and interstrand cross-links. 395(2)

Mutagen and Carcinogen Detection 397(1)

Mutagens can be detected based on their ability to restore mutant gene activity. 397(1)

Direct Reversal of Damage 398(9)

Photolyase reverses damage caused by cyclobutane pyrimidine dimer formation. 398(6)

O6-Alkylguanine, O4-alkylthymine, and phosphotriesters can be repaired by direct alkyl group removal by a suicide enzyme. 404(2)

AlkB catalyzes the oxidative removal of methyl groups in 1-methyladenine and 3-methylcytosine. 406(1)

Base Excision Repair 407(6)

The base excision repair (BER) pathway removes and replaces damaged bases. 407(6)

Nucleotide Excision Repair 413(11)

Nucleotide excision repair removes bulky adducts from DNA by excising an oligonucleotide bearing the lesion and replacing it with new DNA. 413(11)

Mismatch Repair 424(5)

The DNA mismatch repair system removes mismatches and short insertions or deletions that are present in DNA. 424(5)

The SOS Response and Translesion DNA Synthesis 429(12)

Error-prone DNA polymerases catalyze translesion DNA synthesis. 429(1)

RecA and LexA regulate the E. coli SOS response. 429(4)

The SOS signal induces the synthesis of DNA polymerases II, IV, and V. 433(1)

Human cells have at least 14 different template-dependent DNA polymerases. 434(1)

Suggested Reading 435(6)

Recombination 441(63)

Hannah Klein

Introduction to Homologous Recombination 442(4)

Homologous recombination is an essential process for repairing DNA breaks and for ensuring correct chromosome segregation in meiosis. 442(4)

Recombination in Bacteriophage 446(2)

Crossing over involves an exchange of DNA between the two interacting DNA molecules. 446(2)

Recombination in Bacteria 448(1)

Recombination mutants of E. coli have reduced conjugation rates and are sensitive to DNA damage. 448(1)

Early Models of Homologous Recombination 448(11)

The Holliday model of HR proposes a crossed strand intermediate called a Holliday junction. 448(5)

RecA is a strand exchange protein. 453(2)

RecBCD complex prepares double-strand breaks for homologous recombination and alters its activity at chi sites. 455(2)

The Meselson-Radding model of recombination---a second homologous recombination model---is based on one single strand nick for initiation. 457(2)

Transformation in Yeast 459(1)

Yeast repair gapped plasmids by homologous recombination. 459(1)

A Homologous Recombination Model Initiated by a Double-Strand Break 460(1)

The double-strand break repair (DSBR) model is based on a double-strand break for initiation. 460(1)

Eukaryotic Homologous Recombination Proteins 461(7)

Several key homologous recombination proteins are conserved between bacteria and eukaryotes, but there are additional novel proteins found only in eukaryotes. 461(7)

A Variation of the Double-Strand Break Repair Model 468(1)

The synthesis-dependent strand-annealing (SDSA) model is a gene conversion-only model. 468(1)

Meiotic Recombination 469(3)

Some aspects of meiotic recombination are novel. 469(1)

Some recombination proteins are made only in meiotic cells. 469(2)

Meiotic recombination models propose two different types of homologous recombination events. 471(1)

Using Mitotic Recombination to Make Gene Knockouts 472(6)

Mitotic recombination can be used in genetic engineering to make targeted gene disruptions. 472(1)

Gene knockouts in yeast occur by homologous recombination with high efficiency. 473(1)

Gene knockouts in mice also can be made by gene targeting methods. 474(4)

Mitotic Recombination and DNA Replication 478(5)

Mitotic homologous recombination is essential during DNA replication when replication forks collapse. 478(2)

Recombination must be regulated to prevent chromosome rearrangements and genomic instability. 480(2)

The single-strand annealing (SSA) mechanism results in deletions. 482(1)

Repairing a Double-Strand Break without Homology 483(2)

Nonhomologous end-joining is a model for rejoining ends with no homology. 483(2)

Site-Specific Recombination 485(19)

Site-specific recombination occurs at defined DNA sequences and is used for immunoglobulin diversity and by transposaljle elements. 485(1)

Mating type switching in yeast occurs by synthesis-dependent strand-annealing initiated at a defined site. 486(2)

V(D)J recombination produces the immune system diversity. 488(4)

FLP/FRT and Ctt/lox systems can be used to make targeted recombination events. 492(4)

Suggested Reading 496(8)

Transposons and Other Mobile Elements 504(49)

Joseph E. Peters

Transposition 507(31)

The simplest mobile elements in bacteria are called insertion sequences. 507(1)

The transposase forms a specific complex with the ends of the element. 508(2)

Coordinated breakage and joining events occur during transposition. 510(1)

Some elements do cut-and-paste transposition, where the element is directly moved to a new location. 510(2)

Transposition during DNA replication and host DNA repair allow cut-and-paste elements to increase in copy number. 512(1)

Transposons are found at various levels of complexity in bacteria. 513(2)

Replicative transposons leave one copy of the element at the donor site. 515(2)

Transposons in eukaryotes are mechanistically similar to bacteria transposons. 517(2)

Diverse systems allow transposition to be regulated. 519(2)

Most transposons prefer DNA targets that are bent. 521(1)

Transposons can target certain sequences. 521(1)

Some transposons target specific molecular processes. 522(1)

Some elements have evolved the ability to choose between certain target sites. 523(1)

Transposons are important tools for molecular genetics. 524(14)

Conservative Site-Specific Recombination 538(5)

Two families of proteins do conservative site-specific recombination with different pathways. 538(2)

Bacteriophage λ uses a conservative site-specific recombinase to integrate into the host genome. 540(2)

Multiple other systems use the conservative site-specific recombinase reaction. 542(1)

Target-Primed Reverse Transcription 543(10)

Target-primed reverse transcription can mobilize information through an RNA intermediate. 543(1)

Target-primed reverse transcription is used for LINE movement. 544(2)

LINE movement affects genome stability and evolution. 546(2)

Mobile group II introns move by target-primed reverse transcription. 548(1)

Two transesterification reactions allow group II intron movement. 548(1)

Homology to the target site determines if mobile group II introns move by retrotransposition or retro-homing. 549(1)

Suggested Reading 550(3)

SECTION 5 RNA Synthesis and Processing 553(318)

Bacterial RNA Polymerase 554(34)

Introduction to the Bacterial RNA Polymerase 555(4)

RNA polymerase requires a DNA template and four nucleoside triphosphates to synthesize RNA. 555(2)

Bacterial RNA polymerases are large multisubunit proteins. 557(2)

Initiation Stage 559(18)

Bacterial RNA polymerase holoenzyme consists of a core enzyme and sigma factor. 559(1)

A transcription unit must have an initiation signal called a promoter for accurate and efficient transcription to take place. 560(1)

The DNase protection method provides information about promoter DNA. 560(2)

Genetic studies provide additional information about RNA polymerase holoenzyme-promoter interactions. 562(1)

Bacteria have two sigma factor families. 563(2)

The σ54-RNA polymerase requires an activator protein. 565(1)

Footprinting experiments show that σ70RNA polymerase combines with promoter DNA to form a closed and an open complex. 565(3)

Rifampicin binds to RNA polymerase and blocks an early step in RNA synthesis. 568(1)

Core polymerase and σ70RNA polymerase have different conformations. 569(1)

The crystal structure of T. aquaticus core RNA polymerase revealed new details about the way that the enzyme is organized. 570(2)

Crystal structures reveal important information about the way that the open complex is organized. 572(4)

RNA polymerase holoenzyme scrunches DNA during the initial stage of transcription. 576(1)

Transcription Elongation Complex 577(4)

Transcription elongation complex is a highly processive molecular motor. 577(4)

Transcription Termination 581(7)

Bacterial transcription machinery releases RNA strands at intrinsic and Rho-dependent terminators. 581(3)

Suggested Reading 584(4)

Regulation of Bacterial Gene Transcription Messenger RNA 588(64)

Bacterial mRNA may be monocistronic or polycistronic. 589(4)

Bacterial mRNA usually has a short lifetime compared to other kinds of bacterial RNA. 590(1)

Controlling the rate of mRNA synthesis can regulate the flow of genetic information. 591(1)

Gene regulation helps to eliminate wasteful metabolic activity. 591(1)

Messenger RNA synthesis can be controlled by negative and positive regulation. 592(1)

Lactose Operon 593(12)

The E. coli genes lacZ, lacY, and lacA code for β-galactosidase, lactose permease, and β-galactoside transacetylase, respectively. 593(1)

The lac structural genes are regulated. 594(1)

Genetic studies provide information about the regulation of lac mRNA. 595(1)

The operon model explains the regulation of the lactose system. 596(2)

Allolactose is the true inducer of the lactose operon. 598(1)

The Lac repressor binds to the lac operator in vitro. 599(2)

The lac operon has three lac operators. 601(1)

The Lac repressor is a dimer of dimers, where each dimer binds to one lac operator sequence. 601(4)

Catabolite Repression 605(7)

E. coli uses glucose in preference to lactose. 605(1)

The inhibitory effect of glucose on expression of the lac operon is a complicated process. 605(4)

The cAMP CRP complex binds to an activator site (AS) upstream from the lac promoter and activates lac operon transcription. 609(2)

cAMP CRP activates more than 100 operons. 611(1)

Galactose Operon 612(2)

The galactose operon is also regulated by a repressor and cAMP CRP. 612(2)

The AraBAD Operon 614(5)

The AraC activator protein regulates the araBAD operon. 614(5)

Tryptophan Operon 619(6)

The tryptophan (trp) operon is regulated at the levels of transcription initiation, elongation, and termination. 619(6)

Bacteriophage Lambda: A Transcription Regulation Network 625(7)

Lambda phage development is regulated by a complex genetic network. 625(1)

The lytic pathway is controlled by a transcription cascade. 626(2)

The lysogenic pathway is also controlled by a transcription cascade. 628(1)

The CI regulator maintains the lysogenic state. 628(3)

Ultraviolet light induces the λ prophage to enter the lytic pathway. 631(1)

Messenger RNA Degradation 632(3)

Bacterial mRNA molecules are rapidly degraded. 632(3)

Ribosomal RNA and Transfer RNA Synthesis 635(4)

Bacterial ribosomes are made of a large subunit with a 23S and 5S RNA and a small subunit with 16S RNA. 635(1)

E. coli has seven rRNA operons, each coding for a 16S, 23S, and 5S RNA. 636(1)

A promoter upstream element (UP element) increases rrn transcription. 637(1)

Three Fis protein binding sites increase rrn transcription. 638(1)

Regulation of Ribosome Synthesis 639(3)

Amino acid starvation leads the production of guanine nucleotides that inhibit rRNA synthesis. 639(1)

E. coli rRNA and tRNA syntheses increase with growth rate. 640(1)

E. coli regulates r-protein synthesis. 641(1)

Processing rRNA and tRNA 642(10)

Bacteria process the primary transcripts for rRNA and tRNA to form the physiologically active RNA molecules. 642(5)

Suggested Reading 647(5)

RNA Polymerase II: Basal Transcription 652(35)

Introduction to RNA Polymerase II 654(5)

The eukaryotic cell nucleus has three different kinds of RNA polymerase. 654(2)

RNA polymerases I, II, and III can be distinguished by their sensitivities to inhibitors. 656(1)

Each nuclear RNA polymerase has some subunits that are unique to it and some that it shares with the two other nuclear RNA polymerases. 657(2)

RNA Polymerase II Structure 659(6)

High-resolution yeast RNA polymerase II structures help explain how the enzyme works. 659(2)

The crystal structure has been determined for the complete 12-subunit yeast RNA polymerase II bound to a transcription bubble and product RNA. 661(4)

Transcription Initiation Site Identification 665(6)

Nuclear RNA polymerases have limited synthetic capacities. 665(1)

RNA polymerase II transcription begins at specific initiation sites. 665(4)

Several methods can be used to monitor gene expression. 669(2)

RNA Polymerase II Core Promoter 671(2)

The core promoter element extends from 40 bp upstream of the transcription initiation site to 40 bp downstream from this site. 671(2)

General Transcription Factors: Basal Transcription 673(8)

RNA polymerase II requires the assistance of general transcription factors to transcribe naked DNA from specific transcription initiation sites. 673(1)

The core promoter allows a cell-free system to catalyze a low-level of RNA synthesis at the correct transcription initiation site. 673(1)

Pre-initiation complex assembly begins with either TFIID or TATA binding protein (TBP) binding to the core promoter. 674(2)

TFIID can bind to core promoters of protein-coding genes that lack a TATA box. 676(2)

TFIIA or TFIIB bind to the TBP TATA box complex. 678(1)

Sequential binding of RNA polymerase II TFIIF complex, TFIIE, and TFIIH completes pre-initiation complex formation. 679(1)

DNA in the pre-initiation complex is tightly wound around a protein core made of RNA polymerase II and general transcription factors. 680(1)

Transcription Elongation 681(6)

The C-terminal domain of the largest RNA polymerase subunit must be phosphorylated for chain elongation to proceed. 681(1)

A variety of transcription elongation factors help to suppress transient pausing during elongation. 682(1)

Elongation factor SII reactivates arrested RNA polymerase II. 683(1)

The transcription elongation complex is regulated. 683(1)

Suggested Reading 684(3)

RNA Polymerase II: Regulation 687(62)

Regulatory Promoter and Enhancer 688(4)

Linker-scanning mutagenesis reveals the regulatory promoter's presence just upstream from the core promoter. 688(3)

Enhancers can stimulate transcription even when they are located a great distance from the transcription initiation site. 691(1)

The upstream activating sequence (UAS) regulates genes in yeast. 692(1)

Transcription Activator Proteins 692(4)

Transcription activator proteins help to recruit the transcription machinery. 692(2)

A combinatorial process determines gene activity. 694(1)

DNA affinity chromatography can be used to purify transcription activator proteins. 694(2)

A transcription activator protein's ability to stimulate gene transcription can be determined by a transfection assay. 696(1)

DNA-Binding Domains with Helix-Turn-Helix Structures 696(6)

Homeotic genes assign positional identities to cells during embryonic development. 696(4)

Homeotic genes specify transcription activator proteins. 700(1)

The homeodomain contains a helix-turn-helix motif. 700(1)

POU proteins have a homeobox and a POU domain. 701(1)

DNA Binding Domains with Zinc Fingers 702(11)

Many transcription activator proteins have Cys2His2 zinc fingers that bind to DNA in a sequence-specific fashion. 702(3)

Nuclear receptors have Cys4 zinc finger motifs. 705(3)

Ligand-binding domain structure provides considerable information about nuclear receptor function. 708(3)

Gal4, a yeast transcription activator protein belonging to the Cys6 zinc cluster family, regulates the transcription of genes involved in galactose metabolism. 711(2)

DNA Binding Domains with Basic Region Leucine Zippers 713(5)

Basic region leucine zipper (bzip) transcription activator proteins bind to DNA as dimers that are held together through coiled coil interactions. 713(5)

DNA Binding Domains with Helix-Loop-Helix Structures 718(3)

Helix-loop-helix transcription regulatory proteins are dimers. 718(1)

The bHLH zip family of transcription regulators have both HLH and leucine zipper dimerization motifs. 719(2)

Activation Domain 721(9)

The activation domain must associate with a DNA-binding domain to stimulate transcription. 721(1)

Gal4 has DNA binding and activation domains. 722(7)

The yeast two-hybrid assay permits us to detect polypeptides that interact through non-covalent interactions. 729(1)

Mediator 730(4)

Squelching occurs when transcription activator proteins compete for a limiting transcription machinery component. 730(1)

Mediator is required for activated transcription. 731(2)

The yeast Mediator complex associates with the UAS in active yeast genes. 733(1)

Chromatin Modification and Remodeling 734(15)

Chromosomes contain interspersed regions of euchromatin and heterochromatin. 734(1)

Cells remodel or modify chromatin to make its DNA accessible to the transcription machinery. 735(1)

Genetic and biochemical studies led to the discovery of ATP- dependent chromatin remodeling complexes. 736(2)

Histone N-terminal tail acetylation tends to activate transcription. 738(1)

Histone deacetylation tends to lower transcriptional activity. 739(1)

Highly modifiable amino terminal histone tails have combinatorial codes that help to control phenotype. 740(1)

Transcription activators and repressors trigger chromatin remodeling and modification events. 741(3)

Suggested Reading 744(5)

RNA Polymerase II: Co transcriptional and Post-transcriptional Processes 749(78)

Pre-mRNA 750(1)

Eukaryotic cells synthesize large heterogeneous RNA (hnRNA) molecules. 750(1)

Messenger RNA and hnRNA both have poly(A) tails at their 3'-ends. 750(1)

Cap Formation 751(8)

Messenger RNA molecules have 7-methylguanosine caps at their 5'-ends. 751(4)

5'-m7G caps are attached to nascent pre-mRNA chains when the chains are 20 to 30 nucleotides long. 755(1)

All eukaryotes use the same basic pathway to form 5'-m7G caps. 755(2)

CTD must be phosphorylated on Ser-5 to target a transcript for capping. 757(2)

Split Genes 759(1)

Viral studies revealed that some mRNA molecules are formed by splicing pre-mRNA. 759(19)

Amino acid-coding regions within eukaryotic genes may be interrupted by noncoding regions. 760(5)

Exons tend to be conserved during evolution, whereas introns usually are not conserved. 765(3)

A single pre-mRNA can be processed to produce two or more different mRNA molecules. 768(1)

Combinations of the various splicing patterns within individual genes lead to the formation of multiple mRNAs. 768(2)

Most alternatively spliced genes appear to code for proteins that participate in signal transduction. 770(1)

Drosophila form an mRNA that codes for essential protein isoforms by alternative trans-splicing. 771(2)

Pre-mRNA requires specific sequences for precise splicing to occur. 773(1)

Two splicing intermediates resemble PU A lariats. 774(3)

Splicing consists of two coordinated transesterification reactions. 777(1)

Spliceosomes 778(15)

Aberrant antibodies, which are produced by individuals with certain autoimmune diseases, bind to small nuclear ribonucleoprotein particles (snRNPs). 778(1)

snRNPs assemble to form a spliceosome, the splicing machine that excises introns. 778(2)

U1, U2, U4, and U5 snRNPs each contains Sm polypeptides, whereas U6 snRNP contains Sm-like polypeptides. 780(1)

Each U snRNP is formed in a multi-step process. 781(2)

U1, U2, U4, U5, and U6 snRNPs have been isolated as a penta-snRNP in yeast. 783(1)

In vitro studies show that spliceosomes assemble on introns via an ordered pathway. 784(2)

The catalytic site within the spliceosome is probably made of RNA rather than protein. 786(2)

Cells use a variety of mechanisms to regulate splice site selection. 788(4)

Splicing begins as a cotranscriptional process and continues as a posttranscriptional process. 792(1)

Cleavage/Polyadenylation and Transcription Termination 793(11)

Poly(A) tail synthesis and transcription termination are coupled, cotranscriptional processes. 793(7)

Alternative processing forces us to reconsider our concept of the gene. 800(1)

Transcription termination takes place downstream from the poly(A) site. 800(2)

Two models have been proposed to explain RNA polymerase II transcription termination. 802(1)

In higher animals, most histone pre-mRNAs require a special processing mechanism. 803(1)

RNA Editing 804(4)

RNA editing permits a cell to recode genetic information in a systematic and regulated fashion. 804(4)

The human proteome contains a much greater variety of proteins than would be predicted from the human genome. 808(1)

Messenger RNA Export 808(2)

Messenger RNA splicing and export are coupled processes. 808(2)

siRNA and miRNA Pathways for Silencing 810(17)

Short RNA molecules can silence gene expression. 810(5)

The miRNA pathway blocks mRNA translation or causes mRNA degradation. 815(2)

Suggested Reading 817(10)

Ribosomal RNA, Transfer RNA, and Organellar RNA Synthesis 827(44)

Eukaryotic Ribosome 828(1)

The eukaryotic ribosome is made of a small and a large ribonucleoprotein subunit. 828(1)

RNA Polymerase 828(15)

The 5.8S, 18S, and 28S rRNA coding sequences are part of a single transcript. 828(3)

Eukaryotes have multiple copies of rRNA transcription units that are divided into small clusters. 831(1)

Eukaryotic rRNA transcription units are usually arranged in head-to-tail clusters with intergenic spacers between them. 832(1)

The rRNA transcription unit promoter consists of a core promoter and an upstream promoter element (UPE). 832(1)

Ribosomal RNA transcription and processing takes place in the nucleolus. 833(2)

RNA polymerase I is a multisubunit enzyme with a structure similar to that of RNA polymerase II. 835(1)

RNA polymerase I-associated factors are required for transcription. 835(1)

RNA polymerase I also requires two auxiliary transcription factors, upstream binding factor (UBF) and selectivity factor (SL1/TIFIB). 836(2)

The transcription initiation complex can be assembled in vitro by the stepwise addition of individual components. 838(1)

RNA polymerase I acts through a transcription cycle that begins with the formation of a pre-initiation complex. 838(3)

Pre-rRNA undergoes a complex series of cleavages and modifications as it is converted to mature ribosomal rRNAs. 841(2)

Ribozymes and Ribosomal RNA 843(6)

Tetrahymerta thermophila pre-rRNA contains an intron that catalyzes its own excision. 843(6)

Ribosome Assembly 849(3)

Eukaryotic ribosome assembly is a complex multi-step process. 849(3)

RNA Polymerase III 852(10)

RNA polymerase III transcripts are short RNA molecules with a variety of biological functions. 852(2)

RNA polymerase III transcription units have three different types of promoters. 854(1)

The transcription factors required to recruit RNA polymerase III depend on the nature of the promoter. 855(3)

RNA polymerase III does not appear to require additional factors for transcription elongation or termination. 858(1)

Pre-tRNAs require extensive processing to become mature tRNAs. 859(3)

Transcription in Mitochondria 862(3)

Mitochondrial DNA is transcribed to form mRNA, rRNA, and tRNA. 862(3)

Transcription in Chloroplasts 865(6)

Chloroplast DNA is also transcribed to form mRNA, rRNA, and tRNA. 865(2)

Suggested Reading 867(4)

SECTION 6 Protein Synthesis 871(102)

Protein Synthesis: The Genetic Code 872(44)

Discovery of Ribosomes and Transfer RNA 873(2)

Protein synthesis takes place on ribosomes. 873(2)

Transfer RNA 875(12)

An amino acid must be attached to a transfer RNA before it can be incorporated into a protein. 875(3)

All tRNA molecules have CCAOH at their 3'-end. 878(1)

An amino acid attaches to tRNA through an ester bond between the amino acid's carboxyl group and the 2'- or 3'-hydroxyl group on adenosine. 879(2)

The tRNA for alanine was the first naturally occurring nucleic acid to be sequenced. 881(1)

Transfer RNAs have cloverleaf secondary structures. 882(3)

Transfer RNA molecules fold into L-shaped three-dimensional structures. 885(2)

Aminoacyl-tRNA Synthetases 887(9)

Aminoacyl-tRNA synthetases can be divided into two classes, I and II. 887(1)

Some aminoacyl-tRNA synthetases have editing functions. 888(1)

Ile-tRNA synthetase has an editing function. 889(1)

Ile-tRNA synthetase can hydrolyze valyl-tRNAIle and valyl-AMP. 890(1)

A polypeptide insert in the Rossman-fold domain forms the editing site for Ile-tRNA synthetase. 891(1)

Each aminoacyl-tRNA synthetase can distinguish its cognate tRNAs from all other tRNAs. 892(2)

Many gram-positive bacteria and archaeons use an indirect pathway for Gln-tRNA synthesis. 894(1)

Selenocysteine and pyrrolysine are building blocks for polypeptides. 895(1)

Messenger RNA and the Genetic Code 896(20)

Messenger RNA programs ribosomes to synthesize proteins. 896(1)

Three adjacent bases in mRNA that specify an amino acid are called a codon. 897(2)

The discovery that poly(U) directs the synthesis of poly(Phe) was the first step in solving the genetic code. 899(2)

Protein synthesis begins at the amino terminus and ends at the carboxyl terminus. 901(2)

Messenger RNA is read in a 5' to 3' direction. 903(1)

Trinucleotides promote the binding of specific aminoacyl-tRNA molecules to ribosomes. 903(1)

Synthetic messengers with strictly defined base sequences confirmed the genetic code. 904(2)

Three codons, UAA, UAG, and UGA, are polypeptide chain termination signals. 906(1)

The genetic code is nonoverlapping, commaless, almost universal, highly degenerate, and unambiguous. 906(2)

The coding specificity of an aminoacyl-tRNA is determined by the tRNA and not the amino acid 908(2)

Some aminoacyl-tRNA molecules bind to more than one condon because there is some play or wobble in the third base of a codon. 910(1)

The origin of the genetic code remains a puzzle. 911(1)

Suggested Reading 912(4)

Protein Synthesis: The Ribosome 916(57)

Ribosome Structure 917(5)

Bacterial ribosome structure has been determined at atomic resolution. 917(3)

Arachael and eukaryotic ribosome structures appear to be similar to then bacterial ribosome structure. 920(2)

Initiation stage 922(17)

Bacteria, eukaryotes, and archaea each have their own translation initiation pathway. 922(1)

Each bacterial mRNA cistron has its own start codon. 923(1)

Bacteria have in initiator methionine tRNA and an elongator methionine tRNA. 923(2)

The 30S subunit is an obligatory intermediate in polypeptide chain initiation. 925(1)

Initiation factors participate in the formation of 30S and 70S initiation complexes. 925(3)

The mRNA Shine-Dalgarno (SD) sequence interacts with the 16S rRNA anti-Shine-Dalgarno (anti-SD) sequence. 928(2)

Riboswitches regulate translation initiation of some bacterial mRNA molecules. 930(1)

Eukaryotic initiator tRNA is charged with a methionine that is not formylated. 931(1)

Eukaryotic translation initiation proceeds through a scanning mechanism. 932(5)

Eukaryotic have at least twelve different initiation factors. 937(1)

Translation initiation factor phosphorylation regulates protein synthesis in eukaryotes. 937(1)

Translation initiation factor phosphorylation regulates protein synthesis in eukaryotes. 937(1)

The translation initiation pathway in archaea appears to be a mixture of the eukaryotic and bacterial pathways. 938(1)

Elongation stage 939(15)

Polypeptide chain elongation requires three elongation factors. 939(1)

The elongation factors act through a repeating three step cycle. 940(1)

An EFlA GTP aminoacyl-tRNA ternary complex carries the aminoacyl-tRNA to the ribosome. 940(1)

Specific nucleotides in 16S rRNA are essential for sensing the codon-antocodon helix. 940(2)

The EF1A conformation depends on the guanine nucleotide in its GTP binding pocket. 942(2)

EF1B is a GDP-GTP exchange protein. 944(1)

Peptidyl transferase activity resides in the 23S rRNA. 945(6)

The hybrid-states translocation model offers a mechanism for moving tRNA molecules through the ribosome. 951(1)

EF2 GTP stimulates the translocation process. 952(2)

Termination Stage 954(8)

Bacteria have three protein release factors. 954(1)

The class 1 release factors, RF1 and RF2, have one tripeptide that acts as an anticodon and another that binds at the peptidyl transferase site. 954(3)

RF3 is a nonessential G protein that stimulates RF1 or RF2 dissociation from the ribosome. 957(1)

A stalled ribosome translating a truncated mRNA that lacks a termination codon can be rescued by tmRNA. 957(3)

Eukaryotic cells have bacteria-like release factors in their mitochondria and a different kind in their cytoplasm. 960(1)

Mutant tRNA molecules can suppress mutations that create termination codons within a reading frame. 961(1)

Recycling Stage 962(1)

The ribosome release factor (RRF) is required for the bacterial ribosomal complex to disassemble. 962(1)

Signal Sequence 963(10)

The signal sequence plays an important role in directing newly synthesized proteins to specific cellular destinations. 963(3)

Suggested Reading 966(7)
Index 973