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Molecular Biology, 4/e

Molecular Biology, 4/e

Molecular Biology, 4/e

Genes to Proteins

  • By: Burton E. Tropp

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ISBN: 9789380853499

Bind: Paperback

Year: 2012

Pages: 1136

Size: 216 x 279 mm

Publisher: Jones & Bartlett Learning

Published in India by: Jones & Bartlett India

Exclusive Distributors: Viva Books

Sales Territory: India, Nepal, Pakistan, Bangladesh, Sri Lanka


Newly revised, reorganized and updated, Molecular Biology: Genes to ProteinsFourth Edition provides readers with a comprehensive guide through the basic molecular processes and genetic phenomena in both prokaryotic and eurkaryotic cells. Written for the undergraduate and first-year graduate students of molecular biology, molecular genetics, or biochemistry, the text has been updated with the latest data from this ever-changing field. Whenever possible the author uses a discovery approach, urging students to explore the historical and experimental evidence relevant to the important concepts discussed and to examine clues and develop hypotheses that ultimately lead to advances in molecular biology. Divided in to six sections, topics are strategically arranged to cover basic information that is necessary to understand more advanced topics covered in later sections.

New and key features for the Fourth Edition:

  • A new chapter describing small silencing RNAs
  • A revised organization allows instructors to build more easily on critical concepts throughout the course
  • An examination of RNA structure in greater detail than ever before
  • Includes a new description of the ubiquitin proteasome proteolytic pathway
  • The section on DNA sequence has been updated to include the new generation of DNA sequencers
  • Coverage has been added that examines epigenetic programming, which includes a discussion of imprinting and induced pluripotent stem (iPS) cells


Target Audience: 

Written for the undergraduate and first year graduate students within molecular biology or molecular genetics as well as for a professional reference.



Chapter 1: Introduction to Molecular Biology • Intellectual Foundation • Two studies performed in the 1860s provided the intellectual underpinning for molecular biology • Genotypes and Phenotypes • Each gene is responsible for the synthesis of a single polypeptide • Nucleic Acids • Nucleic acids are linear chains of nucleotides • DNA Structure and Function • Transformation experiments led to the discovery that DNA is the hereditary material • Chemical experiments also supported the hypothesis that DNA is the hereditary material • The blender experiment demonstrated that DNA is the genetic material in bacterial viruses • RNA serves as the hereditary material in some viruses • Rosalind Franklin and Maurice Wilkins obtained x-ray diffraction patterns of extended DNA fibers • James Watson and Francis Crick proposed that DNA is a double-stranded helix • The central dogma provides the theoretical framework for molecular biology • Recombinant DNA technology allows us to study complex biological systems • A great deal of molecular biology information is available on the Internet • Suggested reading

SECTION I: Protein Structure and Function

Chapter 2: Protein Structure • The  a-Amino Acids •  a-Amino acids have an amino group and a carboxyl group attached to a central carbon atom • Amino acids are represented by three-letter and one-letter abbreviations • The Peptide Bond •  a-Amino acids are linked by peptide bonds • Protein Purification • Protein mixtures can be fractionated by chromatography. Proteins and other charged biological polymers migrate in an electric field • Primary Structure of Proteins • The amino acid sequence or primary structure of a purified protein can be determined • Polypeptide sequences can be obtained from nucleic acid sequences • The BLAST program compares a new polypeptide sequence with all sequences stored in a data bank • Proteins with just one polypeptide chain have primary, secondary, and tertiary structures while those with two or more chains also have quaternary structures • Weak Noncovalent Bonds • The polypeptide folding pattern is determined by weak noncovalent interactions • Secondary Structures • The  a-helix is a compact structure that is stabilized by hydrogen bonds • The ??-conformation is also stabilized by hydrogen bonds • Loops and turns connect different peptide segments, allowing polypeptide chains to fold back on themselves • Certain combinations of secondary structures, called supersecondary structures or folding motifs, appear in many different proteins • We cannot yet predict secondary structures with absolute certainty • Tertiary Structure • X-ray crystallography and nuclear magnetic resonance studies have revealed the three-dimensional structures of many different proteins • Intrinsically disordered proteins lack an ordered structure under physiological conditions • Structural genomics is a field devoted to solving x-ray and NMR structures in a high throughput manner • The primary structure of a polypeptide determines its tertiary structure • Molecular chaperones help proteins to fold inside the cell • Proteins and Biological Membranes • Proteins interact with lipids in biological membranes • The fluid mosaic model has been proposed to explain the structure of biological membranes • Suggested Reading

Chapter 3: Protein Function • Myoglobin, Hemoglobin, and the Quaternary Structure Concept • Differences in myoglobin and hemoglobin function are explained by differences in myoglobin and hemoglobin structure • Normal adult hemoglobin (HbA) differs from sickle cell hemoglobin (HbS) by only one amino acid • Immunoglobulin G and the Domain Concept • Large polypeptides fold into globular units called domains • Enzymes • Enzymes are proteins that catalyze chemical reactions • Different methods can be used to detect enzyme activity • Enzymes lower the energy of activation but do not affect the equilibrium position • All enzyme reactions proceed through an enzyme-substrate complex • Molecular details for enzyme-substrate complexes have been worked out for many enzymes • Regulatory enzymes control committed steps in biochemical pathways • Regulatory enzymes exhibit sigmoidal kinetics and are stimulated or inhibited by allosteric effectors • Enzyme activity can be altered by covalent modification • G-Protein-Linked Signal Transduction Systems • G-protein-linked signal transduction systems convert extracellular chemical or physical signals into intracellular signals • The Ubiquitin Proteasome Proteolytic Pathway • The ubiquitin proteasome system is responsible for the specific degradation of intracellular proteins • Suggested Reading

SECTION II: Nucleic Acids and Nucleoproteins

Chapter 4: Nucleic Acid Structure • DNA Size and Fragility • DNA molecules vary in size and base composition • DNA molecules are fragile • Recognition Patterns in the Major and Minor Grooves • Enzymes can recognize specific patterns at the edges of the major and minor grooves • DNA Bending • Some base sequences cause DNA to bend • DNA Denaturation and Renaturation • DNA can be denatured • Hydrogen bonds stabilize double-stranded DNA • Base stacking also stabilizes double-stranded DNA • Base stacking is a cooperative interaction • Ionic strength influences DNA structure • The DNA molecule is in a dynamic state • Distant short patches of complementary sequences can base pair in single-stranded DNA • Alkali denatures DNA without breaking phosphodiester bonds • Complementary single strands can anneal to form doublestranded DNA • Helicases • Helicases are motor proteins that use the energy of nucleoside triphosphates to unwind DNA • Single-Stranded DNA Binding Proteins • Single-stranded DNA binding proteins (SSB) stabilize singlestranded DNA • Topoisomers and Topoisomerases • Covalently closed circular DNA molecules can form supercoils • Bacterial DNA usually exists as a covalently closed circle • Plasmid DNA molecules are used to study the properties of circular DNA in vitro • Circular DNA molecules often have superhelical  structures • Supercoiled DNA results from under- or overwinding circular DNA • Superhelices can have single-stranded regions • Topoisomerases catalyze the conversion of one topoisomer into another • Enzymes belonging to the topoisomerase I family can be divided into three subfamilies • Type II topoisomerases require ATP to convert one topoisomer into another • Non-B DNA Conformations • A-DNA is a right-handed double helix with a deep major groove and very shallow minor groove • Z-DNA has a left-handed conformation • DNA conformational changes result from rotation about  single bonds • Several other kinds of non-B DNA structures appear to exist in nature • RNA Structure • RNA performs a wide variety of functions in the cell • RNA secondary structure is dominated by Watson-Crick base pairs • RNA tertiary structures are stabilized by interactions between two or more secondary structure elements • The RNA World Hypothesis • The earliest forms of life on earth may have used RNA as both the genetic material and the biological catalysts needed to maintain life • Suggested Reading

Chapter 5: Techniques in Molecular Biology • Nucleic Acid Isolation • The method used for DNA isolation must be tailored to the organism from which the DNA is to be isolated • Great care must be taken to protect RNA from degradation during its isolation • Different physical techniques are used to study macromolecules • Electron Microscopy • Electron microscopy allows us to see macromolecules • Centrifugal Techniques • Velocity sedimentation can separate macromolecules and provide information about their size and shape • Equilibrium density gradient centrifugation separates particles according to their density • Gel Electrophoresis • Gel electrophoresis separates charged macromolecules by  their rate of migration in an electric field • Gel electrophoresis can be used to separate proteins and determine a polypetide's molecular mass • Capillary gel electrophoresis is a rapid and automated process that provides quantitative data • Pulsed-field gel electrophoresis (PFGE) can separate very large DNA molecules • Nucleases and Restriction Maps • Nucleases are useful tools in DNA investigations • Restriction endonucleases that cleave within specific nucleotide sequences are very useful tools for characterizing DNA • Restriction endonucleases can be used to construct a restriction map of a DNA molecule • Recombinant DNA Technology • DNA fragments can be inserted into plasmid  DNA vectors • Southern blotting is used to detect specific DNA fragments • Northern and Western blotting are used to detect specific RNA and polypeptide molecules, respectively • DNA polymerase I, a multifunctional enzyme with polymerase,  3??5?  exonuclease, and 5??3?  exonuclease activities, can be used to synthesize labeled DNA • DNA polymerase I can synthesize DNA at a nick • The polymerase chain reaction is used to amplify DNA • Site-directed mutagenesis can be used to introduce a specific base change within a gene • DNA Sequence Determination • The Maxam-Gilbert method uses controlled chemical degradation to sequence DNA • The chain termination method for sequencing DNA uses dideoxynucleotides to interrupt DNA synthesis • DNA molecules that are 1 to 8 kbp long can be sequenced by primer walking • DNA sequences can be stitched together by using information obtained from a restriction map • Shotgun sequencing is used to sequence long DNA molecules • The Human Genome Project used hierarchical shotgun assembly to sequence the human genome • Whole genome shotgun sequencing has also been used to sequence the human genome • The human genome sequence provides considerable new  information • A new generation of DNA sequencers provide rapid and  accurate information without the need for electrophoresis or in vivo cloning • Reverse transcriptase can use an RNA molecule as a template  to synthesize DNA • 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 • Suggested Reading

Chapter 6: Chromosome Structure • Bacterial Chromatin • Bacterial DNA is located in the nucleoid • MukB makes an important contribution to the compaction of the bacterial chromosome • Additional nucleoid-associated proteins contribute to bacterial DNA compaction • Mitosis and Meiosis • In higher animals, germ cells have a daploid number of chromosomes and somatic cells have a diploid number • The animal cell life cycle alternates between interphase and mitosis • Mitosis allows cells to maintain the chromosome number • Cohesin is a four subunit complex that keeps sister  chromatids together • Meiosis reduces the chromosome number in half • Karyotype • Chromosome sites are specified according to nomenclature conventions • A karyotype shows an individual cell's metaphase chromosomes arranged in pairs and sorted by size • A great deal of information can be obtained by examining karyotype preparations • Fluorescent in situ hybridization (FISH) provides a great deal of information about chromosomes • The Nucleosome • Five major histone classes interact with DNA in eukaryotic chromatin • The first level of chromatin organization is the nucleosome • X-ray crystallography provides high-resolution images of nucleosome core particles • The precise nature of the interaction between H and the core particle is not known • The 30-nm Fiber • A chain of nucleosomes appears to fold into a 30-nm fiber • The Scaffold Model • The scaffold model was proposed to explain higher order chromatin structure • Condensins and topoisomerase II help to stabilize condensed chromosomes • The Centromere • The centromere is the site of microtubule attachment • The Telomere • The telomere, which is present at either end of a chromosome, is needed for stability • Suggested Reading

SECTION III: Genetics and Virology

Chapter 7: Genetic Analysis in Molecular Biology • Introduction to Genetic Recombination • Genetic recombination involves an exchange of DNA segments between DNA molecules or chromosomes • Recombination frequencies are used to obtain a genetic map • Bacterial Genetics • Bacteria, which are often selected as model systems for genetic analyses, have complex structures • Bacteria can be cultured in liquid or solid media • Specific notations, conventions, and terminology are used in bacterial genetics • Cells with altered genes are called mutants • Some mutants display the mutant phenotype under all conditions, while others display it only under certain conditions • Certain physical and chemical agents are mutagens • Mutants can be classified on the basis of the changes in the DNA • A mutant organism may regain its original phenotype • Mutants have many uses in molecular biology • A genetic test known as complementation can be used to determine the number of genes responsible for a phenotype • E. coli cells can exchange genetic information by conjugation • Approximately 40F factor genes are needed for successful mating and DNA transfer to occur • The F plasmid can integrate into a bacterial chromosome and carry it into a recipient cell • Bacterial mating experiments can be used to produce an E. coli genetic map ?F1 plasmids contain part of the bacterial chromosome • Plasmid replication control functions are usually clustered in a region called the basic replicon • Plasmids often confer advantageous properties to their hosts • Budding Yeast (Saccharomyces cerevisiae) • Yeasts are unicellular eukaryotes • Specific notations, conventions, and terminology are used in yeast genetics • Yeast cells exist in haploid and diploid stages • The yeast mating type is determined by an allele present in the mating type (Mat) locus • Yeast mating factors act as signals to initiate the mating process • Restriction and Amplified Fragment Length Polymorphisms • Recombinant DNA techniques have facilitated genetic analysis in humans and other organisms • Somatic Cell Genetics • Somatic cell genetics can be used to map genes in higher organisms • Animal cells can be studied in culture • Two different animal cells can fuse to form a heterokaryon • Hybrid cells can be used to make monoclonal antibodies • Suggested Reading
Chapter 8: Viruses in Molecular Biology • Introduction to Viruses • Viruses are obligate parasites that can only replicate in a host cell • Introduction to the Bacteriophages • Bacteriophages were of interest because they seemed to have the potential to serve as therapeutic agents to treat bacterial diseases • Investigators belonging to the ?Phage Group? were the first to use viruses as model systems to study fundamental questions about gene structure and function • Bacteriophages come in different sizes and shapes • Bacteriophages have lytic, lysogenic, and chronic life cycles • Bacteriophages form plaques on a bacterial lawn • Bacteria and the phages that infect them are in a constant • struggle for survival • Virulent Bacteriophages • E.coli phage T4 DNA is terminally redundant and circularly  permuted • E. coli phage T7 DNA is terminally redundant but not ?circularly permuted • E. coli phage ?X174 contains a single-stranded circular DNA molecule • Some phages have-single-stranded RNA as their genetic material • Temperate Phages • E. coli phage ?DNA can replicate through a lytic or lysogenic life cycle • E. coli phage P1 can act as generalized transducing particles • Chronic Phages • After infection, a chronic phage programs the host cell for continued virion particle release without killing the cell • Animal Viruses • Polyomaviruses contain circular double-stranded DNA • Adenonviruses have linear blunt-ended, double-stranded DNA with an inverted repeat at each end • Retroviruses use reverse transcriptase to make a DNA copy of their RNA genome • Suggested Reading

SECTION IV: DNA Metabolism

Chapter 9: DNA Replication in Bacteria • General Features of DNA Replication • DNA replication is semiconservative • Bacterial and eukaryotic DNA replication is bidirectional • DNA replication is semidiscontinuous • DNA ligase connects adjacent Okazaki fragments • RNA serves as a primer for Okazaki fragment synthesis • The bacterial replication machinery has been isolated and examined in vitro • Mutant studies provide important information about the enzymes involved in DNA replication • The Initiation Stage • The replicon model proposes that an initiator protein must  bind to a DNA sequence called a replicator at the start of replication • E. coli chromosomal replication begins at oriC • DnaA, the bacterial initiator protein, has four functional  domains • DnaA?ATP assembles to form a filament at oriC, causing the DNA unwinding element (DUE) to melt • DnaB helicases have double-ring structures • DnaC loads DnaB helicase onto the single-stranded DNA generated at the DUE • DnaG (primase) catalyzes RNA primer synthesis • The Elongation Stage • Several enzymes act together at the replication fork • DNA polymerase III is required for bacterial DNA replication • A polymerase's processivity can be determined by using a polymerase ?trap? to bind the polymerase after it dissociates from its DNA substrate • DNA polymerase holoenzyme has ten distinct subunits that form three subassemblies • The core polymerase has one subunit with 5??3?  polymerase activity and another with  3??5?  exonulcease activity • The clamp forms a ring around DNA, tethering the remainder of the polymerase holoenzyme to the DNA • The clamp loader places the sliding clamp around DNA • The DNA polymerase III holoenzyme clamp loader has three ) subunits • The replisome catalyzes coordinated leading and lagging DNA synthesis at the replication fork • Core polymerase is released from the   clamp by a premature release (also called signaling release) or collision release mechanism • Three models have been proposed to explain how helicase moves  5??3?  on the lagging strand while primase moves in the opposite direction as it synthesizes primer • The Termination Stage • Replication terminates when the two growing forks meet in the terminus region, which is located 180?? around the circular chromosome from the origin • The terminus utilization substance (TUS) binds to Ter sites • Regulation of Bacterial DNA Replication • Three mechanisms regulate bacterial DNA replication at the initiation stage • Suggested Reading

Chapter 10: DNA Replication in Eukaryotes and the Archaea • The SV40 DNA Replication System • The SV40 T antigen binds to the origin of replication and unwinds DNA • SV40 T antigen helps to recruit DNA polymerase/ a-primase ?( Pola)  to the proto-replication bubble • Introduction to Eukaryotic DNA Replication • Eukaryotic replication machinery must replicate long linear duplexes with multiple origins of replication • Eukaryotic Replication Initiation • Eukaryotic chromosomes have many replicator sites • Autonomously replicating sequences (ARS) determine the site of DNA chain initiation in yeast • Two-dimensional gel electrophoresis can locate origins of replication • The origin of recognition complex (ORC) serves as the eukaryotic initiator • CDC6 and Cdt1 help load MCM2-7- helicase onto the origin to form a pre-replication complex (pre-RC) • The licensed origin must be activated before replication can take place • Eukaryotic Replication Elongation • Pol • and Pol e are primarily responsible for copying the lagging- and leading-strand templates, respectively • _ The End-Replication Problem • Studies of the Tetrahymena and yeast telomeres suggested that • a terminal transferase-like enzyme is required for telomere formation • Telomerase uses an RNA template to add nucleotide repeats to chromosome ends • Telomerase plays an important role in solving the endreplication problem • Telomerase plays a role in aging and cancer • Replication Coupled Chromatin Synthesis • Chromatin disassembly and reassembly are tightly coupled to  DNA replication • DNA Replication in the Archaea • The archaeal replication machinery is similar to that in eukaryotes • Orc1/Cdc6 recruits MCM to the archaeal origin of replication • The basic steps in archaeal elongation are very similar to those in bacteria and eukaryotes • Suggested Reading

Chapter 11: DNA Damage • Radiation Damage • Ultraviolet light causes cyclobutane pyrimidine dimer (CPD) formation and (6-4) photoproduct formation • X-rays and gamma rays cause many different types of DNA damage • DNA Instability in Water • DNA is damaged by hydrolytic cleavage reactions • Oxidative Damage • Reactive oxygen species damage DNA • Alkylation Damage by Monoadduct Formation • Alkylating agents damage DNA by transferring alkyl groups to centers of negative charge • Many environmental agents must be modified by cell metabolism before they can alkylate DNA • Chemical Cross-Linking Agents • Chemical cross-linking agents block DNA strand separation • Psoralen and related compounds can form monoadducts or cross-links • Cisplatin combines with DNA to form intra- and interstrand cross-links • Mutagen and Carcinogen Detection • Mutagens can be detected based on their ability to restore mutant gene activity • Suggested Reading

Chapter 12: DNA Repair • Direct Reversal of Damage • Photolyase reverses damage caused by cyclobutane pyrimidine dimer formation • O6-Alkylguanine, O4-alkylthymineand phosphotriesters can be repaired by direct alkyl group removal by a suicide enzyme • AlkB catalyzes the oxidative removal of methyl groups in ?1-methyladenine and 3-methylcytosine • Base Excision Repair • The base excision repair (BER) pathway removes and replaces damaged or inappropriate bases • Nucleotide Excision Repair • Nucleotide excision repair removes bulky adducts from DNA by excising an oligonucleotide bearing the lesion and replacing it with new DNA • UvrA, UvrB, and UvrC proteins are required for bacterial nucleotide excision repair • Individuals with the autosomal recessive disease xeroderma  pigmentosum have defects in enzymes that participate in the nucleotide excision repair pathway • Mismatch Repair • The DNA mismatch repair system removes mismatches and short insertions or deletions that are present in DNA • The SOS Response and Translesion DNA Synthesis • Error-prone DNA polymerases catalyze translesion DNA synthesis • RecA and LexA regulate the E. coli SOS response • The SOS signal induces the synthesis of DNA polymerases II, IV, and V • Human cells have at least different template-dependent DNA polymerases • Suggested Reading

Chapter 13: Recombination • Hannah Klein • Introduction to Homologous Recombination • Homologous recombination is an essential process for repairing DNA breaks and for ensuring correct chromosome segregation in meiosis • Early Clues from Bacteriophage • Crossing over involves an exchange of DNA between the two interacting DNA molecules • Early Models of Homologous Recombination • The Holliday model of homologous recombination proposes a crossed strand intermediate called a Holliday junction • The Meselson?Radding model of recombination???a second homologous recombination model???is based on one singlestrand nick for initiation • A Homologous Recombination Model Initiated by a Double-Strand Break • Yeast repair gapped plasmids by homologous recombination • The double-strand break repair (DSBR) model is based on a double-strand break for initiation • Bacterial Homologous Recombination Proteins • E. coli recombination mutants have reduced conjugation rates and are sensitive to DNA damage • RecA is a strand exchange protein • The RecBCD complex prepares double-strand breaks for homologous recombination and alters its activity at chi sites • The RecFOR pathway repairs single-strand gaps • Eukaryotic Homologous Recombination  Proteins • Several key homologous recombination proteins are conserved between bacteria and eukaryotes, but there are additional novel proteins found only in eukaryotes • A Variation of the Double-Strand Break Repair Model • The synthesis-dependent strand-annealing (SDSA) model is a gene conversion-only model • Meiotic Recombination • Some aspects of meiotic recombination are novel • Some recombination proteins are made only in meiotic cells • Meiosis recombination models propose two different types of homologous recombination events • Using Mitotic Recombination to Make Gene Knockouts • Mitotic recombination can be used in genetic engineering to make targeted gene disruptions • Gene knockouts in yeast occur by homologous recombination with high efficiency • Gene knockouts in mice also can be made by gene targeting methods • Mitotic Recombination and DNA Replication • Mitotic homologous recombination is essential during DNA replication when replication forks collapse • Recombination must be regulated to prevent chromosome rearrangements and genomic instability • The single-strand annealing (SSA) mechanism results in deletions • Repairing a Double-Strand Break without Homology • Nonhomologous end-joining is a model for rejoining ends with no homology • Site-Specific Recombination • Site-specific recombination occurs at defined DNA sequences and is used for immunoglobulin diversity and by transposable elements • Mating type switching in yeast occurs by synthesis-dependent strand-annealing initiated at a defined site • V(D)JFRT and Cre/lox systems can be used to make targeted  recombination events •  Suggested Reading

Chapter 14: Transposons and Other Mobile Elements • Joseph E. Peters • Transposition • The simplest mobile elements in bacteria are called insertion sequences • The transposase forms a specific complex with the ends of the mobile element • Coordinated breakage and joining events occur during transposition • Some elements do cut-and-paste transposition, where the element is directly moved to a new location • Transposition during DNA replication and host DNA repair allow cut-and-paste elements to increase in copy number • Transposons are found at various levels of complexity in bacteria • Replicative transposons leave one copy of the element at the donor site • Transposons in eukaryotes are mechanistically similar to bacterial transposons • Diverse systems allow transposition to be regulated • Most transposons prefer DNA targets that are bent • Transposons can target certain sequences • Some transposons target specific molecular processes • Some elements have evolved the ability to choose between certain target sites • Transposons are important tools for molecular genetics • Conservative Site-Specific Recombination • Two families of proteins do conservative site-specific recombination with different pathways • Bacteriophage •  uses a conservative site-specific recombinase to integrate into the host genome • Multiple other systems use the conservative site-specific recombinase reaction • Target-Primed Reverse Transcription • Target-primed reverse transcription can mobilize information through an RNA intermediate • Target-primed reverse transcription is used for LINE movement • LINE movement affects genome stability and evolution • Mobile group II introns move by target-primed reverse transcription • Two transesterification reactions allow group II intron movement • Homology to the target site determines if mobile group II introns move by retrotransposition or retro-homing • Other Mechanisms of DNA Mobilization • Suggested Reading

SECTION V: RNA Metabolism

Chapter 15: Bacterial RNA Polymerase • Introduction to the Bacterial RNA Polymerase Catalyzed Reaction • RNA polymerase requires a DNA template and four nucleoside triphosphates to synthesize RNA • Bacterial RNA polymerases are large multisubunit proteins • Initiation Stage • Bacterial RNA polymerase holoenzyme consists of a coreenzyme and sigma factor • A transcription unit must have an initiation signal called a promoter for accurate and efficient transcription to take place • The DNase protection method provides information about promoter DNA • DNA footprinting shows that • 70 RNA polymerase combines with promoter DNA to form a closed and an open complex • Bacterial RNA polymerase crystal structures show how the enzyme is organized and provide insights into how it works • Genetic and biochemical studies provide additional information about bacterial promoters • Members of the • 70 family have four conserved domains • RNA polymerase scrunches DNA during transcription initiation • Transcription initiation is a stepwise process • Alternative •  factors direct RNA polymerase to genes that code for proteins that bacteria require to survive under specific types of environmental stress The ?54-RNA polymerase requires an activator protein • Transcription Elongation Complex • The transcription elongation complex is a highly processive molecular motor • The trigger loop and the ??? bridge helix help to move RNA polymerase forward by one nucleotide during each nucleotide addition cycle • Pauses influence the overall transcription elongation rate • RNA polymerase can detect and remove incorrectly incorporated nucleotides • Transcription Termination • Bacterial transcription machinery releases RNA strands at intrinsic and Rho-dependent terminators • Antibiotics that Target Bacterial RNA • Polymerase • RNA polymerase is a target for broad spectrum antibacterial therapy • Suggested Reading

Chapter 16:  Regulation of Bacterial Gene Transcription • Messenger RNA • Bacterial mRNA may be monocistronic or polycistronic • Bacterial mRNA usually has a short lifetime compared to other kinds of bacterial RNA • Controlling the rate of mRNA synthesis can regulate the flow of genetic information • Messenger RNA synthesis can be controlled by negative and positive regulation • Lactose Operon • The E. coli genes lacZ, lacY, and lacA code for ??-galactosidase,  lactose permease, and ??-galactoside transacetylase, respectively • The lac structural genes are regulated • Genetic studies provide information about the regulation of lac mRNA • The operon model explains the regulation of the lactose system • Allolactose is the true inducer of the lactose operon • The Lac repressor binds to the lac operator in vitro • The lac operon has three lac operators • The Lac repressor is a dimer of dimers, where each dimer binds to one lac operator sequence • Catabolite Repression • E. coli uses glucose in preference to lactose • The inhibitory effect of glucose on expression of the lac operon is a complicated process • The cAMP • CRP complex binds to an activator site (AS)  upstream from the lac promoter and activates lac operon transcription • cAMP • CRP activates more than 100 operons • Galactose Operon • The galactose operon is also regulated by a repressor and cAMP?CRP • The araBAD Operon • The AraC activator protein regulates the araBAD operon • Tryptophan Operon • The tryptophan (trp) operon is regulated at the levels of transcription initiation, elongation, and termination • Bacteriophage Lambda: A Transcription Regulation • Network • Phage •  development is regulated by a complex genetic network • The lytic pathway is controlled by a transcription cascade • The lysogenic pathway is also controlled by a transcription cascade • The CI regulator maintains the lysogenic state • Ultraviolet light induces the ?? prophage to enter the lytic pathway • Messenger RNA Degradation • Bacterial mRNA molecules are rapidly degraded • Ribosomal RNA and Transfer RNA Synthesis • Bacterial ribosomes are made of a large subunit with a 23S and 5S RNA and a small subunit with 16S RNA • E. coli has seven rRNA operons, each coding for a 16S, 23S,  and 5S RNA • A promoter upstream element (UP element) increases rrn transcription • Three Fis protein binding sites increase rrn transcription • Regulation of Ribosome Synthesis • Amino acid starvation leads to the production of guanine nucleotides that inhibit rRNA synthesis • E. coli rRNA and tRNA syntheses increase with  growth rate • E. coli regulates r-protein synthesis • Processing rRNA and tRNA • Bacteria process the primary transcripts for rRNA and tRNA to form the physiologically active RNA molecules • Suggested Reading

Chapter 17: RNA Polymerase II: Basal Transcription • Introduction to RNA Polymerase II • The eukaryotic cell nucleus has three different kinds of RNA polymeraseII • RNA polymerases I, II, and III can be distinguished by their sensitivities to inhibitors • Each nuclear RNA polymerase has some subunits that are unique to it and some that it shares with the two other nuclear RNA polymerases • RNA Polymerase II Structure • High-resolution yeast RNA polymerase II structures help explain how the enzyme works • The crystal structure has been determined for the complete 12-subunit yeast RNA polymerase II bound to a transcription bubble and product RNA • Transcription Start Site Identification • Nuclear RNA polymerases have limited synthetic capacities • Various techniques have been devised to locate RNA  polymerase II transcription start sites • Cap analysis of gene expression (CAGE) is a high throughput technique used to identify transcription start sites and their flanking promoters • The Core Promoter • The core promoter extends from  40 bp upstream of the transcription start site to 40 bp downstream from this site • General Transcription Factors: Basal Transcription • RNA polymerase II requires the assistance of general transcription factors to transcribe naked DNA from specific transcription start sites • The core promoter allows a cell-free system to catalyze a lowlevel of RNA synthesis at the correct transcription start site • When the core promoter has a TATA box, preinitiation complex assembly begins with either TFIID or TATA binding protein (TBP) binding to the core promoter • TFIID can bind to core promoters of protein-coding genes that lack a TATA box • TFIIA is not required to reconstitute the minimum transcription system • TFIIB helps to convert the closed promoter to an open promoter • Sequential binding of RNA polymerase II?TFIIF complex, TFIIE, and TFIIH completes preinitiation complex  formation • Transcription Elongation • The C-terminal domain of the largest RNA polymerase subunit must be phosphorylated for chain elongation to proceed • A variety of transcription elongation factors help to suppress transient pausing during elongation • Elongation factor SII reactivates arrested RNA polymerase II • The transcription elongation complex is regulated • Archaeal RNA Polymerase • The archaea have a single RNA polymerase that is similar to RNA polymerase II • Suggested Reading

Chapter 18: RNA Polymerase II: Regulation • Regulatory Promoters, Enhancers, and Silencers • Linker-scanning mutagenesis reveals the regulatory promoter's presence just upstream from the core promoter • Enhancers stimulate transcription and silencers block transcription • The upstream activating sequence (UAS) regulates genes in yeast • Transcription Activator Proteins • Transcription activator proteins help to recruit the transcription machinery • A combinatorial process determines gene activity • DNA affinity chromatography can be used to purify transcription activator proteins • A transcription activator protein's ability to stimulate gene transcription can be determined by a transfection assay • DNA-Binding Domains with Helix-Turn-Helix Structures • Homeotic genes assign positional identities to cells during embryonic development • Homeotic genes specify transcription activator proteins • The homeodomain contains a helix-turn-helix motif • POU proteins have a homeobox and a POU domain • DNA Binding Domains with Zinc Fingers • Many transcription activator proteins have Cys2His2 zinc fingers that bind to DNA in a sequence-specific  fashion • Nuclear receptors have Cys4 zinc finger motifs • Ligand-binding domain structure provides considerable information about nuclear receptor function • Gal 4 a yeast transcription activator protein belonging to the Cys6 zinc cluster family, regulates the transcription of genes involved in galactose metabolism • Loop-Sheet-Helix DNA Binding Domain •  p53 has a loop-sheet-helix DNA binding domain • DNA Binding Domains with Basic Region Leucine  Zippers • Basic region leucine zipper (bzip) transcription activator proteins bind to DNA as dimers that are held together through coiled coil interactions • DNA-Binding Domains with Helix-Loop-Helix  Structures • Helix-loop-helix transcription regulatory proteins are dimers • The bHLH zip family of transcription regulators have both HLH and leucine zipper dimerization motifs • Activation Domain • The activation domain must associate with a DNA-binding domain to stimulate transcription • Gal4 has DNA binding and activation domains • The yeast two-hybrid assay permits us to detect polypeptides that interact through noncovalent interactions • Mediator • Squelching occurs when transcription activator proteins compete for a limiting transcription machinery component • Mediator is required for activated transcription • The yeast Mediator complex associates with the UAS in active yeast genes • Epigenetic Modifications • Cells remodel or modify chromatin to make the DNA in chromatin accessible to the transcription machinery • DNA methylation plays an important role in determining whether chromatin will be silenced or actively expressed in vertebrates • Epigenetics is the study of inherited changes in phenotype caused by changes in chromatin other than changes in DNA sequence • Genomic imprinting in mammals determines whether a maternal or paternal gene will be expressed • Pluripotent cells usually become more specialized during development • A cell nucleus from a terminally differentiated cell can be reprogrammed by an enucleated egg cell to produce a live  animal • Lineage restricted cells can be programmed to produce induced pluripotent stem (iPS) cells • Suggested Reading

Chapter 19: RNA Polymerase II: Cotranscriptional and Posttranscriptional Processes • Pre-mRNA • Eukaryotic cells synthesize large heterogeneous RNA  (hnRNA) molecules • Messenger RNA and hnRNA both have poly(A) tails at their 3?-ends • Cap Formation • Messenger RNA molecules have 7-methylguanosine caps at  their 5?-ends ?5?-m7G caps are attached to nascent pre-mRNA chains when the chains are 20 to 30 nucleotides long • All eukaryotes use the same basic pathway to form 5?-m7G caps • CTD must be phosphorylated on Ser-5 to target a transcript for capping • Split Genes • Viral studies revealed that some mRNA molecules are formed by splicing pre-mRNA • Amino acid?coding regions within eukaryotic genes may be interrupted by noncoding regions • Exons tend to be conserved during evolution, whereas introns usually are not conserved • A single pre-mRNA can be processed to produce two or more different mRNA molecules • Combinations of the various splicing patterns within individual genes lead to the formation of multiple mRNAs • Drosophila form an mRNA that codes for essential protein isoforms by alternative trans-splicing • Pre-mRNA requires specific sequences for precise splicing to occur • Two splicing intermediates resemble lariats • Splicing consists of two coordinated transesterification reactions • Spliceosomes • Aberrant antibodies, which are produced by individuals with certain autoimmune diseases, bind to small nuclear  ribonucleoprotein particles (snRNPs) • snRNPs assemble to form a spliceosome, the splicing machine that excises introns • U1, U2, U4, and U5 snRNPs each contains Sm polypeptides, whereas U6 snRNP contains Sm-like polypeptides • Each U snRNP is formed in a multistep process • U1, U2, U4, U5, and U6 snRNPs have been isolated as a penta-snRNP in yeast • In vitro studies show that spliceosomes assemble on introns via an ordered pathway • RNA and protein may both contribute to the spliceosome's catalytic site • Cells use a variety of mechanisms to regulate splice site selection • Splicing begins as a cotranscriptional process and continues as a posttranscriptional process • Cleavage/Polyadenylation and Transcription Termination • Poly(A) tail synthesis and transcription termination are coupled, cotranscriptional processes • Transcription units often have two or more alternate polyadenylation sites • Alternative processing forces us to reconsider our concept of the gene • Transcription termination takes place downstream from the poly(A) site • RNA polymerase II transcription termination appears to involve allosteric changes and a 5??3?  exonuclease • In higher animals, most histone pre-mRNAs require a special processing mechanism • RNA Editing • RNA editing permits a cell to recode genetic information in a systematic and regulated fashion • The human proteome contains a much greater variety of proteins than would be predicted from the human genome • Messenger RNA Export • Messenger RNA splicing and export are coupled processes •  Suggested Reading

Chapter 20: RNA Polymerases I and III and Organellar RNA Polymerases • Eukaryotic Ribosome • The eukaryotic ribosome is made of a small and a large ribonucleoprotein subunit • RNA Polymerase I • The 5.8S, 18S, and 28S rRNA coding sequences are part of a single transcript • Eukaryotes have multiple copies of rRNA transcription units arranged in clusters on just a few chromosomes • The rRNA transcription unit promoter consists of a core promoter and an upstream promoter element (UPE) • Ribosomal RNA transcription and processing takes place in the nucleolus • RNA polymerase I is a multisubunit enzyme with a structure similar to that of RNA polymerase II • RNA polymerase I?associated factors are required for transcription • RNA polymerase I requires two auxiliary transcription factors, upstream binding factor (UBF) and selectivity factor (SL1/ TIF-B) • The transcription initiation complex can be assembled in vitro by the stepwise addition of individual components • RNA polymerase I acts through a transcription cycle that begins with the formation of a pre-initiation complex • Pre-rRNA undergoes a complex series of cleavages and modifications as it is converted to mature ribosomal • rRNAs • Self-Splicing Ribosomal RNA • Tetrahymena thermophila pre-rRNA contains an intron that catalyzes its own excision • Ribosome Assembly • Eukaryotic ribosome assembly is a complex multistep process • RNA Polymerase III • RNA polymerase III transcripts are short RNA molecules with a variety of biological functions • RNA polymerase III transcription units have three different types of promoters • The transcription factors required to recruit RNA polymerase III depend on the nature of the promoter • RNA polymerase III does not appear to require additional factors for transcription elongation or termination • Pre-tRNAs require extensive processing to become mature  tRNAs • Transcription in Mitochondria • Mitochondrial DNA is transcribed to form mRNA, rRNA, and  tRNA • Transcription in Chloroplasts • Chloroplast DNA is also transcribed to form mRNA, rRNA, and tRNA • Suggested Reading

Chapter 21: Small Silencing RNAs • RNA Interference (RNAi) Triggered by Exogenous • Double-Stranded RNA • The nematode worm Caenorhabditis elegans is an attractive organism for molecular biology studies RNA interference was discovered in the nematode worm  Caenorhabditis elegans • In vitro studies helped to elucidate the RNA interference pathway • Dicer acts as a molecular ruler that generates double-stranded • RNA fragments of discrete size • The guide strand's 5?-phosphate and 3?-ends bind to sites in the Argonaute protein's Mid and PAZ domains, respectively • RISC loading complex is required for siRISC formation • RNAi blocks virus replication and prevents transposon activation • Transitive RNAi • In some organisms, RNA interference that starts at one site spreads throughout the entire organism • SID-, an integral membrane protein in C. elegans, assists in the systemic spreading of the silencing signal • ERI-, a 3?? 5 exonuclease in C. elegans, appears to be a negative regulator of RNA interference • RNAi as an Investigational Tool •  RNAi is a powerful tool for investigating functional genomics • The MicroRNA Pathway • The miRNA pathway blocks mRNA translation or causes mRNA degradation • Piwi Interacting RNAs (piRNAs) • piRNAs help to maintain germline stability in animals • Endogenous siRNA • Animals have a functional endogenous siRNA pathway • Heterochromatin Assembly • The RNAi machinery can establish and maintain heterochromatin • Suggested Reading

SECTION VI: Protein Synthesis

Chapter 22: Protein Synthesis: The Genetic Code • Discovery of Ribosomes and Transfer RNA • Protein synthesis takes place on ribosomes • Transfer RNA • An amino acid must be attached to a transfer RNA before it can be incorporated into a protein • All tRNA molecules have CCAOH at their 3?-ends • 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 • The tRNA for alanine was the first naturally occurring nucleic acid to be sequenced • Transfer RNAs have cloverleaf secondary structures • Transfer RNA molecules fold into L-shaped three-dimensional structures • Aminoacyl-tRNA Synthetases • Aminoacyl-tRNA synthetases can be divided into two classes, I and II • Some aminoacyl-tRNA synthetases have editing functions • Ile-tRNA synthetase can hydrolyze valyl-tRNAIle and valyl-  AMP? A polypeptide insert in the Rossman-fold domain forms the editing site for Ile-tRNA synthetase • Editing-defective alanyl-tRNA synthetase causes a neurodegenerative disease in mice • Each aminoacyl-tRNA synthetase can distinguish its cognate tRNAs from all other tRNAs • Many gram-positive bacteria and archaea use an indirect pathway for Gln-tRNA synthesis • Selenocysteine and pyrrolysine are building blocks for polypeptides • Messenger RNA and the Genetic Code • Messenger RNA programs ribosomes to synthesize proteins • Three adjacent bases in mRNA that specify an amino acid are called a codon • The discovery that poly(U) directs the synthesis of poly(Phe) was the first step in solving the genetic code • Protein synthesis begins at the amino terminus and ends at the carboxyl terminus • Messenger RNA is read in a 5? to 3? direction • Trinucleotides promote the binding of specific aminoacyltRNA molecules to ribosomes • Synthetic messengers with strictly defined base sequences confirmed the genetic code • Three codons, UAA, UAG, and UGA, are polypeptide chain termination signals • The genetic code is nonoverlapping, commaless, almost universal, highly degenerate, and unambiguous • The coding specificity of an aminoacyl-tRNA is determined by the tRNA and not the amino acid • Some aminoacyl-tRNA molecules bind to more than one codon because there is some play or wobble in the third base of a codon • The origin of the genetic code remains a puzzle • Suggested Reading

Chapter 23: Protein Synthesis: The Ribosome • Ribosome Structure • Bacterial ribosome structure has been determined at atomic resolution • Arachael and eukaryotic ribosome structures appear to be similar to the bacterial ribosome structure • Initiation Stage in Bacteria • Protein synthesis can be divided into four stages • Bacteria, eukaryotes, and archaea each have their own translation initiation pathway • Each bacterial mRNA open reading frame has its own start codon • Bacteria have an initiator methionine tRNA and an elongator methionine tRNA • The  30S subunit is an obligatory intermediate in polypeptide chain initiation • Initiation factors participate in the formation of 30S and 70S initiation complexes • The mRNA Shine-Dalgarno (SD) sequence interacts with the 16S rRNA anti-Shine-Dalgarno (anti-SD) sequence • Riboswitches regulate translation initiation of some bacterial • mRNA molecules • Initiation Stage in Eukaryotes • Eukaryotic initiator tRNA is charged with a methionine that is not formylated • Eukaryotic translation initiation proceeds through a scanning mechanism • Eukaryotes have at least twelve different initiation factors • Translation initiation factor phosphorylation regulates protein synthesis in eukaryotes • The translation initiation pathway in archaea appears to be a mixture of the eukaryotic and bacterial pathways • Elongation Stage • Polypeptide chain elongation requires elongation factors • The elongation factors act through a repeating cycle • An EF-Tu?GTP?aminoacyl-tRNA ternary complex carries the aminoacyl-tRNA to the ribosome • An additional elongation factor, EF-P, is required to synthesize the first peptide bond • Specific nucleotides in S rRNA are essential for sensing the codon-anticodon helix • EF-Ts is a GDP-GTP exchange protein • The ribosome is a ribozyme • The hybrid-states translocation model offers a mechanism for moving tRNA molecules through the ribosome • EF-G?GTP stimulates the translocation process • Termination Stage in Bacteria • Bacteria have three protein release factors • The class release factors, RF1 and RF2, have one tripeptide that acts as an anticodon and another that binds at the peptidyl transferase center • RF3 is a nonessential G protein that stimulates RF 1or RF2 dissociation from the ribosome • A stalled ribosome translating a truncated mRNA that lacks a termination codon can be rescued by tmRNA • Mutant tRNA molecules can suppress mutations that create termination codons within a reading frame • Termination Stage in Eukaryotes • Eukaryotic cells have bacteria-like release factors in their mitochondria and a different kind in their cytoplasm • Recycling Stage • The ribosome recycling factor (RRF) is required for the bacterial ribosomal complex to disassemble • Nascent Polypeptide Processing and Folding • Ribosomes have associated enzymes that process nascent polypeptides and chaperones that help to fold the nascent polypeptides • Signal Sequence • The signal sequence plays an important role in directing newly synthesized proteins to specific cellular destinations • Suggested Reading • INDEX


About the Author:

Burton E. Tropp-Queens College/CUNY
Burton Tropp received a Ph.D. in Biochemistry and Molecular Biology from Harvard University. After completing his graduate research on the mechanism of methylation of transfer RNA he studied protein synthesis in regenerating rat liver while a post-doctoral fellow in the Department of Microbiology and Immunology at the Harvard Medical School. He then joined the faculty of the City University of New York where he is currently a professor in the Department of Chemistry and Biochemistry at Queens College and in the Ph.D. Biochemistry Program at the City University of New York Graduate Center. He teaches biochemistry and biochemical genetics at both the undergraduate and graduate levels. His major research interest has been the genetic aspects of lipid metabolism. He has authored more than 50 peer-reviewed papers in this research area.


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