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Biology Sylvia S. Mader Michael Windelspecht Chapter 12 Molecular Biology of the Gene Lecture Outline See separate FlexArt PowerPoint slides for all figures and tables pre-inserted into PowerPoint without notes. 1 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 12.1 The Genetic Material • Frederick Griffith investigated virulence of Streptococcus pneumoniae Concluded that virulence could be passed from a dead strain to a nonvirulent living strain Transformation • Further research by Avery et al. Discovered that DNA is the transforming substance DNA from dead cells was being incorporated into the genome of living cells 2 The Genetic Material • Griffith’s Transformation Experiment Mice were injected with two strains of pneumococcus: an encapsulated (S) strain and a non-encapsulated (R) strain. • The S strain is virulent (the mice died); it has a mucous capsule and forms “shiny” colonies. • The R strain is not virulent (the mice lived); it has no capsule and forms “dull” colonies. 3 Griffith’s Transformation Experiment Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. capsule Injected live R strain has no capsule and mice do not die. Injected live S strain has capsule and causes mice to die. a. b. Injected heatkilled S strain does not cause mice to die. c. Injected heat-killed S strain plus live R strain causes mice to die. Live S strain is withdrawn from dead mice. d. 4 The Genetic Material • Transformation of organisms today: Result is the so-called genetically modified organisms (GMOs) • Invaluable tool in modern biotechnology today • Commercial products that are currently much used • Green fluorescent protein (GFP) can be used as a marker – A jellyfish gene codes for GFP – The jellyfish gene is isolated and then transferred to a bacterium, or the embryo of a plant, pig, or mouse. – When this gene is transferred to another organism, the organism glows in the dark 5 Transformation of Organisms 6 The Genetic Material • DNA contains: Two Nucleotides with purine bases • Adenine (A) • Guanine (G) Two Nucleotides with pyrimidine bases • Thymine (T) • Cytosine (C) 7 The Genetic Material • Chargaff’s Rules: The amounts of A, T, G, and C in DNA: • Are constant among members of the same species • Vary from species to species In each species, there are equal amounts of: • A and T • G and C All this suggests that DNA uses complementary base pairing to store genetic information Each human chromosome contains, on average, about 140 million base pairs The number of possible nucleotide sequences is 4140,000,000 8 Nucleotide Composition of DNA Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. NH2 adenine (A) C N C HC C CH N O HO P O O 5 4 CH2 N C thymine (T) N O HN nitrogen-containing base CH C O CH3 C N O O C H H C 3 OH HO H C1 C H 2 H P O C C HN 4 CH2 O C H H C1 C H 2 H sugar = deoxyribose NH2 N O HO P C O O 5 4 CH2 O N CH C CH N N O O C H H C 3 OH a. Purine nucleotides C cytosine (C) N CH H2N C phosphate 5 H C 3 OH O guanine (G) O HO H C1 C H 2 H P O b. Pyrimidine nucleotides O 5 4 CH2 O C H H C 3 OH H C1 C H 2 H DNA Composition in Various Species (%) Species A T G C Homo sapiens (human) 31.0 31.5 19.1 18.4 Drosophila melanogaster (fruit fly) Zea mays (corn) Neurospora crassa (fungus) 27.3 25.6 23.0 27.6 25.3 23.3 22.5 24.5 27.1 22.5 24.6 26.6 Escherichia coli (bacterium) Bacillus subtilis (bacterium) 24.6 28.4 24.3 29.0 25.5 21.0 25.6 21.6 c. Chargaff’s data 9 The Genetic Material • X-Ray diffraction: Rosalind Franklin studied the structure of DNA using X-rays. She found that if a concentrated, viscous solution of DNA is made, it can be separated into fibers. Under the right conditions, the fibers can produce an X-ray diffraction pattern • She produced X-ray diffraction photographs. • This provided evidence that DNA had the following features: – DNA is a helix. – Some portion of the helix is repeated. 10 X-Ray Diffraction of DNA Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Rosalind Franklin diffraction pattern diffracted X-rays a. X-ray beam Crystalline DNA b. c. © Photo Researchers, Inc.; c: © Science Source/Photo Researchers, Inc. 11 The Genetic Material • The Watson and Crick Model (1953) Double helix model is similar to a twisted ladder • Sugar-phosphate backbones make up the sides • Hydrogen-bonded bases make up the rungs Complementary base pairing ensures that a purine is always bonded to a pyrimidine (A with T, G with C) Received a Nobel Prize in 1962 12 Watson and Crick Model of DNA Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 3.4 nm 0.34 nm 2 nm b. d. C a. G 5′ end sugar-phosphate backbone T 3′ end P A C G S P S A T P P T S 3′ end A P S G P 5′ end C P complementary base pairing c. C G P sugar hydrogen bonds a: © Photodisk Red/Getty RF; d: © A. Barrington Brown/Photo Researchers 13 12.2 Replication of DNA • DNA replication is the process of copying a DNA molecule. • Semiconservative replication - each strand of the original double helix (parental molecule) serves as a template (mold or model) for a new strand in a daughter molecule. 14 Semiconservative Replication Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 5′ 3′ G G C C A A region of parental DNA double helix T C G T A G A A DNA polymerase enzyme G G C C T G A region of replication: new nucleotides are pairing with those of parental strands region of completed replication new strand old strand daughter DNA double helix 5′ old strand 3′ new strand daughter DNA double helix 15 Replication of DNA • Replication requires the following steps: Unwinding, or separation of the two strands of the parental DNA molecule Complementary base pairing between a new nucleotide and a nucleotide on the template strand Joining of nucleotides to form the new strand • Each daughter DNA molecule contains one old strand and one new strand 16 Replication of DNA • Prokaryotic Replication Bacteria have a single circular loop of DNA Replication moves around the circular DNA molecule in both directions Produces two identical circles The process begins at the origin of replication Replication takes about 40 minutes, but the cell divides every 20 minutes • A new round of replication can begin before the previous round is completed 17 Replication of DNA • Eukaryotic Replication DNA replication begins at numerous points along each linear chromosome DNA unwinds and unzips into two strands Each old strand of DNA serves as a template for a new strand Complementary base-pairing forms a new strand paired with each old strand • Requires enzyme DNA polymerase 18 Replication of DNA • Eukaryotic Replication Replication bubbles spread bidirectionally until they meet The complementary nucleotides are joined to form new strands. Each daughter DNA molecule contains an old strand and a new strand. Replication is semiconservative: • One original strand is conserved in each daughter molecule, i.e., each daughter double helix has one parental strand and one new strand. 19 Replication of DNA • Accuracy of Replication DNA polymerase is very accurate, yet makes a mistake about once per 100,000 base pairs. • Capable of identifying and correcting errors 20 12.3 The Genetic Code of Life • Genes Specify Enzymes Beadle and Tatum: • Experiments on the fungus Neurospora crassa • Proposed that each gene specifies the synthesis of one enzyme • One-gene-one-enzyme hypothesis 21 The Genetic Code of Life • The mechanism of gene expression DNA in genes specify information, but information is not structure and function Genetic information is expressed into structure and function through protein synthesis • The expression of genetic information into structure and function: DNA in a gene determines the sequence of nucleotides in an RNA molecule RNA controls the primary structure of a protein 22 The Central Dogma of Molecular Biology Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. nontemplate strand 5 DNA 3 A G C G A C C C C T C G C T G G G G 3 5 template strand transcription in nucleus 5 mRN A translation at ribosome 3 A G C codon 1 G A N C codon 2 O polypeptide C C C C codon 3 O N C C O N C C R1 R2 R3 Serine Aspartate Proline 23 The Genetic Code of Life • RNA is a polymer of RNA nucleotides RNA nucleotides contain the sugar ribose instead of deoxyribose RNA nucleotides are of four types: uracil (U), adenine (A), cytosine (C), and guanine(G) Uracil (U) replaces thymine (T) of DNA • Types of RNA • Messenger (mRNA) - Takes a message from DNA in the nucleus to ribosomes in the cytoplasm • Ribosomal (rRNA) - Makes up ribosomes, which read the message in mRNA • Transfer (tRNA) - Transfers the appropriate amino acid to the ribosomes for protein synthesis 24 Structure of RNA Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 5′ end G P U S A base is uracil instead of thymine P C S P S P S ribose 3′ end one nucleotide 25 RNA Structure Compared to DNA structure 26 The Genetic Code of Life • The unit of the genetic code consists of codons, each of which is a unique arrangement of symbols • Each of the 20 amino acids found in proteins is uniquely specified by one or more codons The symbols used by the genetic code are the mRNA bases • Function as “letters” of the genetic alphabet • Genetic alphabet has only four “letters” (U, A, C, G) Codons in the genetic code are all three bases (symbols) long • Function as “words” of genetic information • Permutations: – There are 64 possible arrangements of four symbols taken three at a time – Often referred to as triplets • Genetic language only has 64 “words” 27 Messenger RNA Codons Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. First Base U C A G Second Base Third Base U C A G UUU phenylalanine UCU serine UAU tyrosine UGU cysteine U UUC phenylalanine UCC serine UAC tyrosine UGU cysteine C UUA leucine UCA serine UAA stop UGA stop A UUG leucine CUU leucine UCG serine UAG stop UGG tryptophan G CCU proline CAU histidine CGU arginine U CUC leucine CCC proline CAC histidine CGC arginine C CUA leucine CCA proline CAA glutamine CGA arginine A CUG leucine CCG proline CAG glutamine CGG arginine G AUU isoleucine AUC isoleucine ACU threonine ACC threonine AAU asparagine AAC asparagine AGU serine AGC serine U AUA isoleucine ACA threonine AAA lysine AGA arginine A AUG (start) methionine ACG threonine AAG lysine AGG arginine G GUU valine GCU alanine GAU aspartate GGU glycine U GUC valine GCC alanine GAC aspartate GGC glycine C GUA valine GUG valine GCA alanine GCG alanine GAA glutamate GAG glutamate GGA glycine GGG glycine A C 28 G The Genetic Code of Life • Properties of the genetic code: Universal • With few exceptions, all organisms use the code the same way • Encode the same 20 amino acids with the same 64 triplets Degenerate (redundant) • There are 64 codons available for 20 amino acids • Most amino acids encoded by two or more codons Unambiguous (codons are exclusive) • None of the codons code for two or more amino acids • Each codon specifies only one of the 20 amino acids Contains start and stop signals • Punctuation codons • Like the capital letter we use to signify the beginning of a sentence, and the period to signify the end 29 12.4 First Step: Transcription • Transcription A segment of DNA serves as a template for the production of an RNA molecule The gene unzips and exposes unpaired bases Serves as template for mRNA formation Loose RNA nucleotides bind to exposed DNA bases using the C=G and A=U rule When entire gene is transcribed into mRNA, the result is a pre-mRNA transcript of the gene The base sequence in the pre-mRNA is complementary to the base sequence in DNA 30 First Step: Transcription • A single chromosome consists of one very long molecule encoding hundreds or thousands of genes • The genetic information in a gene describes the amino acid sequence of a protein The information is in the base sequence of one side (the “sense” strand) of the DNA molecule The gene is the functional equivalent of a “sentence” • The segment of DNA corresponding to a gene is unzipped to expose the bases of the sense strand The genetic information in the gene is transcribed (rewritten) into an mRNA molecule The exposed bases in the DNA determine the sequence in which the RNA bases will be connected together RNA polymerase connects the loose RNA nucleotides together • The completed transcript contains the information from the gene, but in a mirror image, or complementary form 31 Transcription Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 3′ 5′ terminator template strand gene strand C 3′ direction of polymerase movement T RNA polymerase DNA template strand mRNA transcript promoter 5′ 32 5′ 3′ to RNA processing RNA Polymerase Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 200m a. spliceosome DNA RNA polymerase RNA transcripts b. © Oscar L. Miller/Photo Researchers, Inc. 33 First Step: Transcription • Pre-mRNA is modified before leaving the eukaryotic nucleus. Modifications to the ends of the primary transcript: • Cap on the 5′ end – The cap is a modified guanine (G) nucleotide – Helps a ribosome determine where to attach when translation begins • Poly-A tail of 150-200 adenines on the 3′ end – Facilitates the transport of mRNA out of the nucleus – Inhibits degradation of mRNA by hydrolytic enzymes. 34 First Step: Transcription • Pre-mRNA, is composed of exons and introns. The exons will be expressed, The introns, occur in between the exons. • Allows a cell to pick and choose which exons will go into a particular mRNA RNA splicing: • Primary transcript consists of: – Some segments that will not be expressed (introns) – Segments that will be expressed (exons) • Performed by spliceosome complexes in nucleoplasm – Introns are excised – Remaining exons are spliced back together • Result is a mature mRNA transcript 35 First Step: Transcription • In prokaryotes, introns are removed by “self-splicing”—that is, the intron itself has the capability of enzymatically splicing itself out of a pre-mRNA 36 Messenger RNA Processing in Eukaryotes Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. exon exon exon DNA intron intron transcription exon exon exon pre-mRNA 5 intron exon 3 intron exon exon 3 poly-A tail 5 intron cap intron spliceosome exon exon exon 3 poly-A tail 5 cap pre-mRNA splicing intron RNA mRNA 5 cap 3 poly-A tail nuclear pore in nuclear envelope nucleus cytoplasm 37 First Step: Transcription • Functions of introns: As organismal complexity increases; • The number of protein-coding genes does not keep pace • But the proportion of the genome that is introns increases Possible functions of introns: • Exons might combine in various combinations – Would allow different mRNAs to result from one segment of DNA • Introns might regulate gene expression • Introns may encourage crossing-over during meiosis Exciting new picture of the genome is emerging 38 12.5 Second Step: Translation • Translation The sequence of codons in the mRNA at a ribosome directs the sequence of amino acids into a polypeptide A nucleic acid sequence is translated into a protein sequence • tRNA molecules have two binding sites: One associates with the mRNA transcript The other associates with a specific amino acid Each of the 20 amino acids in proteins associates with one or more of 64 types of tRNA • Translation An mRNA transcript associates with the rRNA of a ribosome in the cytoplasm or a ribosome associated with the rough endoplasmic reticulum The ribosome “reads” the information in the transcript Ribosome directs various types of tRNA to bring in their specific amino acid “fares” The tRNA specified is determined by the code being translated in the mRNA transcript 39 Second Step: Translation • tRNA molecules come in 64 different kinds • All are very similar except that One end bears a specific triplet (of the 64 possible) called the anticodon The other end binds with a specific amino acid type tRNA synthetases attach the correct amino acid to the correct tRNA molecule • All tRNA molecules with a specific anticodon will always bind with the same amino acid 40 Structure of a tRNA Molecule Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. amino acid leucine 3’ 5’ hydrogen bonding amino acid end anticodon G C A G U C A C U anticodon end A U C C U C mRNA 5’ 3’ codon a. b. 41 Second Step: Translation • Ribosomes Ribosomal RNA (rRNA): • Produced from a DNA template in the nucleolus of a nucleus • Combined with proteins into large and small ribosomal subunits A completed ribosome has three binding sites to facilitate pairing between tRNA and mRNA • The E (for exit) site • The P (for peptide) site, and • The A (for amino acid) site 42 Ribosome Structure and Function Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. large subunit 3 5 mRNA tRNA binding sites small subunit b. Binding sites of ribosome a. Structure of a ribosome outgoing tRNA polypeptide incoming tRNA mRNA c. Function of ribosomes d. Polyribosome Courtesy Alexander Rich 43 Second Step: Translation • Initiation: Components necessary for initiation are: • • • • • Small ribosomal subunit mRNA transcript Initiator tRNA, and Large ribosomal subunit Initiation factors (special proteins that bring the above together) Initiator tRNA: • Always has the UAC anticodon • Always carries the amino acid methionine • Capable of binding to the P site 44 Second Step: Translation • Small ribosomal subunit attaches to mRNA transcript Beginning of transcript always has the START codon (AUG) • Initiator tRNA (UAC) attaches to P site • Large ribosomal subunit joins the small subunit 45 Initiation Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. amino acid methionine Met initiator tRNA 5 E site mRNA 3 P site A site Met small ribosomal subunit large ribosomal subunit U AC AUG 5 A small ribosomal subunit binds to mRNA; an initiator tRNA pairs with the mRNA start codon AUG. start codon 3 The large ribosomal subunit completes the ribosome. Initiator tRNA occupies the P site. The A site is ready for the next tRNA. 46 Initiation Second Step: Elongation • “Elongation” refers to the growth in length of the polypeptide • RNA molecules bring their amino acid fares to the ribosome Ribosome reads a codon in the mRNA • Allows only one type of tRNA to bring its amino acid • Must have the anticodon complementary to the mRNA codon being read • The incoming tRNA joins the ribosome at its A site Methionine of the initiator tRNA is connected to the amino acid of the 2nd tRNA by a peptide47 bond Second Step: Elongation • The second tRNA moves to P site (translocation) • The spent initiator tRNA moves to the E site and exits • The ribosome reads the next codon in the mRNA Allows only one type of tRNA to bring its amino acid • Must have the anticodon complementary to the mRNA codon being read • Joins the ribosome at its A site The dipeptide on the 2nd amino acid is connected to the amino acid of the 3rd tRNA by a peptide bond 48 Elongation Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Met peptide Ser bond tRNA Trp 3 A tRNA–amino acid approaches the ribosome and binds at the A site. C A U G U A 3 2 Two tRNAs can be at a ribosome at one time; the anticodons are paired to the codons. Val Asp Asp C U G G A C 5 Trp peptide bond Val Asp C A U G U A G A C 5 1 Trp Val Val Ala Ala Trp C U G C U G G A C G U A 3 5 3 Thr Ser Ser Ala anticodon C A U G U A Met Ser C U G Ala Met Met asp Peptide bond formation attaches the peptide chain to the newly arrived amino acid. 4 G A C A C C 3 5 The ribosome moves forward; the “empty” tRNA exits from the E site; the next amino acid–tRNA complex is approaching the ribosome. Elongation 49 Second Step: Translation • Termination: Previous tRNA moves to the P site Spent tRNA moves to the E site and exits Ribosome reads the STOP codon at the end of the mRNA • UAA, UAG, or UGA • Does not code for an amino acid A protein called a release factor binds to the stop codon and cleaves the polypeptide from the last tRNA The ribosome releases the mRNA and dissociates into subunits The same mRNA may be read by another ribosome 50 Termination Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Trp release factor Val Trp Val U U A A U G A stop codon 5′ 3′ The ribosome comes to a stop codon on the mRNA. A release factor binds to the site. 3′ 5′ The release factor hydrolyzes the bond between the last tRNA at the P site and the polypeptide, releasing them. The ribosomal subunits dissociate. Termination 51 Summary of Protein Synthesis in Eukaryotes Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. TRANSCRIPTION TRANSLATION 1. DNA in nucleus serves as a template for mRNA. DNA 2. mRNA is processed before leaving the nucleus. introns large and small ribosomal subunits mRNA 5 3. mRNA moves into cytoplasm and becomes associated with ribosomes. pre-mRNA 3 mRNA amino 4. tRNAs with anticodons carry amino acids to mRNA. acids nuclear pore peptide ribosome 5 tRNA U A C A UG 3 anticodon codon 5. During initiation, anticodon-codon complementary base pairing begins as the ribosomal subunits come together at a start codon. 5 CCC UGG UU U GGG AC C AA A G 3 6. During elongation, polypeptide synthesis takes place one amino acid at a time. 8. During termination, a ribosome reaches a stop codon; mRNA and ribosomal subunits disband. 7. Ribosome attaches to rough ER. Polypeptide enters lumen, where it folds and is modified. 52 12.6 Structure of the Eukaryotic Chromosome • Each chromosome contains a single linear DNA molecule, but is composed of more than 50% protein. • Some of these proteins are concerned with DNA and RNA synthesis • Histones play primarily a structural role The most abundant proteins in chromosomes Five primary types of histone molecules Responsible for packaging the DNA • The DNA double helix is wound at intervals around a core of eight histone molecules (called a nucleosome) • Nucleosomes are joined by “linker” DNA. 53 Structure of Eukaryotic Chromosomes Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 2nm DNA double helix 11 nm 1. Wrapping of DNA around histone proteins. a. Nucleosomes (“beads on a string”) histones nucleosome 2. Formation of a three-dimensional zigzag structure via histone H1 and other DNA-binding proteins. histone H1 b. 30-nm fiber 30 nm 300 nm 3. Loose coiling into radial loops . c. Radial loop domains euchromatin 700 nm 4. Tight compaction of radial loops to form heterochromatin. d. Heterochromatin 5. Metaphase chromosome forms with the help of a protein scaffold. 1,400 nm e. Metaphase chromosome a: © Ada L. Olins and Donald E. Olins/Biological Photo Service; b: Courtesy Dr. Jerome Rattner, Cell Biology and Anatomy, University of Calgary; c: Courtesy of Ulrich Laemmli and J.R. Paulson, Dept. of Molecular Biology, University of Geneva, Switzerland; d: © Peter Engelhardt/Department of Pathology and Virology, Haartman Institute/Centre of Excellence in Computational Complex Systems Research, Biomedical Engineering and Computational Science, Faculty of Information and Natural Sciences, Helsinki University of Technology, Helsinki, Finland 54