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Chapter 10 Molecular Biology of the Gene PowerPoint Lectures for Biology: Concepts and Connections, Fifth Edition – Campbell, Reece, Taylor, and Simon Lectures by Chris Romero Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Sabotage Inside Our Cells • Viruses are biological saboteurs – Hijack the genetic material of host cells in order to reproduce themselves – May remain permanently dormant in the body • Viruses share some characteristics of living organisms but are not generally considered alive – Genetic material composed of nucleic acid – Not cellular – Cannot reproduce on their own • First understanding of DNA based on viruses Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings THE STRUCTURE OF THE GENETIC MATERIAL 10.1 Experiments showed that DNA is the genetic material • Hershey-Chase experiments in 1952 determined that the heredity material was DNA not protein – Studied the simple bacteriophage T2 – Showed that the virus injects its DNA into host cells and reprograms them to produce more viruses Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings LE 10-1a Head DNA Tail 300,000 Tail fiber LE 10-1b Phage Radioactive protein Bacterium DNA Batch 1 Radioactive protein Mix radioactively labeled phages with bacteria. The phages infect the bacterial cells. Batch 2 Radioactive DNA Empty protein shell Radioactivity in liquid Phage DNA Centrifuge Agitate in a blender to separate phages outside the bacteria from the cells and their contents. Pellet Centrifuge the mixture Measure the so bacteria form a radioactivity in pellet at the bottom of the pellet and the test tube. the liquid. Radioactive DNA Centrifuge Pellet Radioactivity in pellet LE 10-1c Phage attaches to bacterial cell. Phage injects DNA. Phage DNA directs host cell to make more phage DNA and protein parts. New phages assemble. Cell lyses and releases new phages. 10.2 DNA and RNA are polymers of nucleotides • Nucleic acids are polynucleotides made of long chains of nucleotide monomers Single-ring pyrimidines: thymine (T), cytosine ( C) Double-ring purines: adenine (A), guanine (G) Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings LE 10-2c Nitrogenous base (A, G, C, or U) Phosphate group Uracil (U) Sugar (ribose) LE 10-2b Cytosine (C) Thymine (T) Pyrimidines Adenine (A) Guanine (G) Purines LE 10-2a Sugar-phosphate backbone Phosphate group A Nitrogenous base Sugar A C C DNA nucleotide Nitrogenous base (A, G, C, or T) Phosphate group T T Thymine (T) G G Sugar (deoxyribose) T T DNA nucleotide DNA polynucleotide • DNA and RNA are identical except for two things – Nitrogenous bases • DNA: A, C, G, T • RNA: A, G, C, U – Sugars • DNA: Deoxyribose • RNA: Ribose Animation: DNA and RNA Structure Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings LE 10-2d Key Hydrogen atom Carbon atom Nitrogen atom Oxygen atom Phosphorus atom 10.3 DNA is a double-stranded helix • James Watson and Francis Crick worked out the three-dimensional structure of DNA, based on X-ray crystallography by Rosalind Franklin & Maurice Wilkins Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings • DNA consists of two polynucleotide strands wrapped around each other in a double helix – Sugar-phosphate backbones are on the outside and nitrogenous bases on the inside – Each base pairs with a complementary partner • A with T, and G with C – Hydrogen bonds between the bases hold the strands together Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings LE 10-3c Twist Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings LE 10-3d C G T A T A Base pair C Hydrogen bond T G C G A T A C G C G T T C A G A A T A T A G A Ribbon model T C T Partial chemical structure Computer model DNA REPLICATION 10.4 DNA replication depends on specific base pairing • Replication process – DNA strands separate – Enzymes use each strand as a template to assemble new nucleotides into complementary strands • The mechanism of DNA replication is semiconservative – Each new double helix consists of one old and one new strand Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings LE 10-4a A T A T C G C G G C G A T A T A T Parental molecule of DNA A C C Nucleotides Both parental strands serve as templates T A T A T G C G C G C G C G C T A T A T A T A T A Two identical daughter molecules of DNA LE 10-4b G C A T G C C G A T C Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings 10.5 DNA replication: A closer look • DNA replication begins at specific sites (origins of replication) on the double helix – Proteins (helicase) attach and break the hydrogen bonds separating the strands – Replication proceeds in both directions, creating replication bubbles – A second strand of new DNA is synthesized along each separated strand by DNA polymerases, which position free nucleotides across from complementary nucleotides Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings DNA Replication • Parent strands open, daughter strands elongate – Replication occurs simultaneously at many sites Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings LE 10-5a Origin of replication Parental strand Daughter strand Bubble Two daughter DNA molecules • DNA's sugar-phosphate backbones are oriented in opposite directions (anti-parallel) – The enzyme DNA polymerase adds nucleotides at only the 3’ end • One daughter strand is synthesized as a continuous piece • The other strand is synthesized as a series of short pieces (Okazaki Fragments) • The two strands are connected by the enzyme DNA ligase Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings LE 10-5b 3 end 5 end P HO 5 2 4 3 1 A T 2 P C G P P G C P P T 3 end 4 5 P OH 3 1 A P 5 end LE 10-5c DNA polymerase molecule 3 5 5 Daughter strand synthesized continuously Parental DNA 3 3 5 5 3 DNA ligase Overall direction of replication Daughter strand synthesized In pieces Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings THE FLOW OF GENETIC INFORMATION FROM DNA TO RNA TO PROTEIN 10.6 The DNA genotype is expressed as proteins, which provide the molecular basis for phenotypic traits • The information constituting an organism's genotype is carried in its sequence of DNA bases • A particular gene—a linear sequence of many nucleotides—specifies a particular polypeptide Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings How Did Scientists Discover That Genes Are Made of DNA? – Transformed Bacteria Revealed the Link Between Genes and DNA Genes Are Made of DNA • Known since the late 1800s: 1. Heritable information is carried in discrete units called genes 2. Genes are parts of structures called chromosomes 3. Chromosomes are made of deoxyribonucleic acid (DNA) and protein Genes Are Made of DNA • Transformed bacteria revealed the link between genes and DNA Genes Are Made of DNA • F. Griffith worked with two strains of Streptococcus pneumoniae bacteria – S strain caused pneumonia when injected into mice, killing them – R strain did not cause pneumonia when injected Genes Are Made of DNA • Griffith made a sample of heat-killed S strain and mixed it with R strain – Injection of combination into mice caused pneumonia and death Genes Are Made of DNA • Deductions from Griffith’s experiment (1920s) – Living safe bacteria (R strain) were changed by something in the dead (but normally diseasecausing) S strain – The living R strain bacteria were transformed by genetic material released by the S strain Genes Are Made of DNA • Later findings by Avery, MacLeod, and McCarty (1940s) – The transforming molecule from the S strain was DNA The Link Between DNA and Protein • • DNA contains the molecular blueprint of every cell Proteins are the “molecular workers” of the cell The Link Between DNA and Protein • • Proteins control cell shape, function, reproduction, and synthesis of biomolecules The information in DNA genes must therefore be linked to the proteins that run the cell One Gene Encodes One Protein • Synthesis of new molecules inside the cell occurs through biochemical pathways • Each step in a biochemical pathway is catalyzed by a protein enzyme One Gene Encodes One Protein • George Beadle and Edward Tatum showed that one DNA gene encodes the information for one enzyme (protein) in a biochemical pathway • Studies of inherited metabolic disorders in mold suggested that phenotype is expressed through proteins • The hypothesis has been restated to one gene-one polypeptide RNA Intermediaries • DNA holds the information on how to make proteins • DNA in eukaryotes is kept in the nucleus; it can’t leave • Protein synthesis occurs at ribosomes in the cytoplasm • How do instructions on DNA get to the ribosomes outside of the nucleus? RNA Intermediaries • DNA information must be carried by an intermediary (RNA) from nucleus to cytoplasm RNA Intermediaries • RNA differs structurally from DNA – RNA is single stranded – RNA uses the sugar ribose – RNA uses the nitrogenous base uracil (U) instead of thymine (T) RNA Intermediaries • There are three types of RNA involved in protein synthesis – Messenger RNA (mRNA) carries DNA gene information to the ribosome – Transfer RNA (tRNA) brings amino acids to the ribosome – Ribosomal RNA (rRNA) is part of the structure of ribosomes Transcription and Translation • DNA directs protein synthesis in a two-step process 1. Information in a DNA gene is copied into mRNA in the process of transcription 2. mRNA, together with tRNA, amino acids, and a ribosome, synthesize a protein in the process of translation 10.7 Genetic information written in codons is translated into amino acid sequences • Genetic information flows from DNA RNA protein • Nucleotide monomers represent letters in an alphabet that can form words in a language – Triplet code • Three-letter words (codons) • Each word codes for one amino acid in a polypeptide Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings LE 10-7a DNA molecule Gene 1 Gene 2 Gene 3 DNA strand A A A C C G G C A A A A U U U G G C C G U U U U Transcription RNA Codon Translation Polypeptide Amino acid 10.8 The genetic code is the Rosetta stone of life • The genetic code specifies the correspondence between RNA codons and amino acids in proteins – Includes start (AUG-methionine) and 3 stop codons – Redundant but not ambiguous • Nearly all organisms use exactly the same genetic code Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings LE 10-8a Second base C U A UAU UCU UUU Phe U UGU Cys Tyr UAC UCC UUC G C UGC Ser U UUA UCA UAA Stop UGA Stop A UUG UCG UAG Stop UGG Trp G CUU CCU CAU CUC CCC CAC Leu U CGU His Leu C CUA CGU Pro CCA C Arg CAA CGA A CGG G Gln CUG CCG CAG AUU ACU AAU AUC lle ACC U AGU Ser Asn AAC AGC C ACA AAA AGA A ACG AAG AGG G GCU GAU GGU U Thr A AUA AUG Met or start GUU Arg Lys Asp GUC G GCC Val GUA GAC GCA C GGC Gly Ala GAA GGA A GGG G Glu GUG GCG GAG Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings LE 10-8b Strand to be transcribed T A C T T C A A A A T C A T G A A G T T T T A G U A G DNA Transcription A U G A A G U U U RNA Stop codon Start codon Translation Polypeptide Met Lys Phe 10.9 Transcription produces genetic messages in the form of RNA • One DNA strand serves as a template for the new RNA strand • RNA polymerase constructs the RNA strand in a multistep process – Initiation: • RNA polymerase attaches to the promotor • Synthesis starts Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings • Elongation: – RNA synthesis continues – RNA strand peels away from DNA template – DNA strands come back together in transcribed region • Termination: – RNA polymerase reaches a terminator sequence at the end of the gene – Polymerase detaches Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings LE 10-9a RNA nucleotides RNA polymerase C C A A U C C A T A G G T Direction of transcription Newly made RNA A T Template strand of DNA LE 10-9b RNA polymerase DNA of gene Promoter DNA Terminator DNA Initiation Elongation Termination Completed RNA Area shown In Figure 10.9A Growing RNA RNA polymerase 10.10 Eukaryotic RNA is processed before leaving the nucleus • The RNA that encodes an amino acid sequence is messenger RNA (mRNA) • In prokaryotes, transcription and translation both occur in the cytoplasm Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings • In eukaryotes, RNA transcribed in the nucleus is processed before moving to the cytoplasm for translation • RNA Splicing – Noncoding segments called introns are cut out – Remaining exons are joined to form a continuous coding sequence – A cap and a tail are added to the ends Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings LE 10-10 Exon Intron Exon Intron Exon DNA Cap RNA transcript with cap and tail Transcription Addition of cap and tail Introns removed Tail Exons spliced together mRNA Coding sequence Nucleus Cytoplasm 10.11 Transfer RNA molecules serve as interpreters during translation • Transfer RNA (tRNA) molecules match the right amino acid to the correct codon • tRNA is a twisted and folded single strand of RNA – Anticodon loop at one end recognizes a particular mRNA codon by base pairing – Amino acid attachment site is at the other end • Each amino acid is joined to the correct tRNA by a specific enzyme Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings LE 10-11a Amino acid attachment site Hydrogen bond RNA polynucleotide chain Anticodon LE 10-11b Amino acid attachment site Anticodon 10.12 Ribosomes build polypeptides • A ribosome consists of two subunits – Each is made up of proteins and ribosomal RNA (rRNA) • The subunits of a ribosome – Hold the tRNA and mRNA close together in binding sites during translation – Allow amino acids to be connected into a polypeptide chain Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings LE 10-12a tRNA molecules Growing polypeptide Large subunit mRNA Small subunit LE 10-12b tRNA-binding sites Large subunit mRNA binding site Small subunit LE 10-12c Next amino acid to be added to polypeptide Growing polypeptide tRNA mRNA Codons 10.13 An initiation codon marks the start of an mRNA message • The initiation phase of translation – Brings together mRNA, a specific tRNA, and the two subunits of a ribosome – Establishes exactly where translation will begin • Ensures that mRNA codes are translated in the correct sequence Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings • Initiation is a two-step process – Step 1 • mRNA binds to a small ribosomal subunit • Initiator tRNA, carrying the amino acid Met, binds to the start codon – Step 2 • A large ribosomal subunit binds to the small one, forming a functional ribosome • Initiator tRNA fits into one binding site; the other is vacant for the next tRNA Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings LE 10-13a Start of genetic message End LE 10-13b Large Ribosomal subunit Initiator tRNA P site U A C A U G U A C A UG Start codon mRNA A site Small ribosomal subunit 10.14 Elongation adds amino acids to the polypeptide chain until a stop codon terminates translation • Once initiation is complete, amino acids are added one by one in a three-step elongation process 1. Codon recognition 2. Peptide bond formation 3. Translocation • Elongation continues until a stop codon reaches the ribosome's A site, terminating translation Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings LE 10-14 Amino acid Polypeptide A site P site Anticodon mRNA Codons Codon recognition mRNA movement Stop codon Peptide bond formation New peptide bond Translocation Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings LE 10-15 Transcription DNA mRNA RNA polymerase mRNA is transcribed from a DNA template. Translation Amino acid Enzyme Each amino acid attaches to its proper tRNA with the help of a specific enzyme and ATP. ATP tRNA Anticodon Initiator tRNA U AC AU G Start Codon mRNA Large Initiation of ribosomal polypeptide synthesis subunit The mRNA, the first tRNA, and the ribosomal Sub units come together. Small ribosomal subunit New peptide bond forming Growing polypeptide Codons mRNA Elongation A succession of tRNAs add their amino acids to the polypeptide chain as the mRNA is moved through the ribosome, one codon at a time. Polypeptide Termination Stop codon The ribosome recognizes a stop codon. The polypeptide is terminated and released. 10.15 Review: The flow of genetic information in the cell is DNA RNA protein • The sequence of codons in DNA, via the sequence of codons in RNA, spells out the primary structure of a polypeptide 1. Transcription of mRNA from a DNA template 2. Attachment of amino acid to tRNA 3. Initiation of polypeptide synthesis 4. Elongation 5. Termination Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings 10.16 Mutations can change the meaning of genes • Mutation: any change in the nucleotide sequence of DNA – Caused by errors in DNA replication or recombination, or by mutagens – Can involve large regions of a chromosome or a single base pair – Can cause many genetic diseases, such as sickle-cell disease Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Mutations Fuel Evolution • Mutations are heritable changes in the DNA • Approx. 1 in 105-106 eggs or sperm carry a mutation • Most mutations are harmful or neutral Mutations Fuel Evolution • Mutations create new gene sequences and are the ultimate source of genetic variation • Mutant gene sequences that are beneficial may spread through a population and become common LE 10-16a Normal hemoglobin DNA C T Mutant hemoglobin DNA T mRNA C A T G U A mRNA G A A Normal hemoglobin Sickle-cell hemoglobin Glu Val • Two general categories of genetic mutations – Base substitutions replace one base with another • Most are harmful but may occasionally have no effect or be beneficial – Base insertions or deletions alter the reading frame • Result is most likely a nonfunctioning polypeptide • Mutagenesis caused by spontaneous error or a physical or chemical mutagen Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings LE 10-16b Normal gene A U G A A G U U U G G C G C A mRNA Protein Met Lys Phe Gly Ala Base substitution A U G A Met A G U Lys G A Phe Base deletion A U U C G Ser C A Ala U Missing U Met G A A Lys G U U Leu G G C Ala G C A His U MICROBIAL GENETICS 10.17 Viral DNA may become part of the host chromosome • Viruses are infectious particles consisting of nucleic acid enclosed in a protein capsid • Viruses depend on their host cells for the replication, transcription, and translation of their nucleic acid – DNA enters host bacterium, circularizes, and enters one of two pathways Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings – Lytic cycle • Host produces more viruses • Host cell lyses (breaks open) to release new viruses Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings – Lysogenic cycle • Phage DNA inserted by recombination into the host chromosome; is now a prophage • Prophages replicated each time host cell divides; passed on to generations of daughter cells • Does not destroy host • Environmental signal may trigger switch from lysogenic to lytic cycle Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings LE 10-17 Phage Attaches to cell Phage DNA Cell lyses, releasing phages Bacterial chromosome Phage injects DNA Many cell divisions Lytic cycle Phages assemble Lysogenic cycle Phage DNA circularizes Prophage Lysogenic bacterium reproduces normally, replicating the prophage at each cell division OR New phage DNA and proteins are synthesized Phage DNA inserts into the bacterial chromosome by recombination CONNECTION 10.18 Many viruses cause disease in animals • Structure of a virus that invades animal cells – Genetic material may be RNA (examples: flu, HIV) or DNA (examples: hepatitis, herpes) – Protein coat – Sometimes a membranous envelope with glycoprotein spikes • The envelope helps the virus enter and leave the host cell during its reproductive cycle Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings LE 10-18a Membranous envelope RNA Protein coat Glycoprotein spike LE 10-18b VIRUS Viral RNA (genome) Plasma membrane of host cell Glycoprotein spike Protein coat Envelope Entry Uncoating Viral RNA (genome) RNA synthesis by viral enzyme Protein synthesis RNA synthesis (other strand) mRNA Template New viral genome New viral proteins Assembly Exit CONNECTION 10.19 Plant viruses are serious agricultural pests • Most plant viruses – Have RNA genomes – Enter their hosts via wounds in the plant's outer layers – May spread throughout the plant through plasmodesmata • There is no cure for most plant viruses Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings LE 10-19 Protein RNA CONNECTION 10.20 Emerging viruses threaten human health • Emerging viruses have appeared suddenly or have recently come to the attention of scientists – Examples: HIV, SARS, Ebola, West Nile • Processes contributing to emergence – Mutation – Contact between species – Spread from isolated populations Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings 10.21 The AIDS virus makes DNA on an RNA template • HIV, the AIDS virus, is a retrovirus – Flow of genetic information is RNA _ DNA – Inside a cell, HIV uses its RNA as a template for making DNA – The enzyme reverse transcriptase catalyzes reverse transcription Animation: HIV Reproductive Cycle Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings LE 10-21a Envelope Glycoprotein Protein coat RNA (two identical strands) Reverse transcriptase LE 10-21b Viral RNA CYTOPLASM NUCLEUS DNA strand Chromosomal DNA Doublestranded DNA Viral RNA and proteins Provirus DNA RNA 10.22 Bacteria can transfer DNA in three ways • Bacteria can transfer genes from cell to cell by one of three processes – Transformation: the uptake of foreign DNA from the surrounding environment – Transduction: transfer of bacterial genes by a phage – Conjugation: union of two bacterial cells and the transfer of DNA between them Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings LE 10-22a DNA enters cell Fragment of DNA from another bacterial cell Bacterial chromosome (DNA) LE 10-22b Phage Fragment of DNA from another bacterial cell (former phage host) LE 10-22c Mating bridge Sex pili Donor cell (“male”) Recipient cell (“female”) • Once new DNA is in a bacterial cell, part of it may integrate into the recipient's chromosome – Occurs by crossing over between the two molecules – Leaves the recipient with a recombinant chromosome Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings LE 10-22d Donated DNA Recipient cell’s chromosome Crossovers Degraded DNA Recombinant chromosome 10.23 Bacterial plasmids can serve as carriers for gene transfer • The F factor is a piece of bacterial DNA – Carries genes for things needed for conjugation – Contains an origin of replication – Can transfer chromosomal DNA by integrating into the donor bacterium's chromosome or entering the cell as a plasmid Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings LE 10-23a F factor (integrated) Male (donor) cell Origin of F replication Bacterial chromosome F factor starts replication and transfer of chromosome Recipient cell Only part of the chromosome transfers Recombination can occur LE 10-23b F factor (plasmid) Male (donor) cell Bacterial chromosome F factor starts replication and transfer Plasmid completes transfer and circularizes Cell now male • Plasmids – Small circular DNA molecules separate from the bacterial chromosome – Can serve as carriers for the transfer of genes Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Colorized TEM 2,000 LE 10-23c Plasmids