Molecular Biology of the Gene

Transcription

1 hapter 12: pp BIOLOY 10th Edition Molecular Biology of the ene 2 nm opyright The Mcraw-Hill ompanies, Inc. Permission required for reproduction or display. b nm 3.4 nm T sugar-phosphate backbone Sylvia S. Mader P A T A P P P Sugar hydrogen bonds PowerPoint Lecture Slides are prepared by Dr. Isaac Barjis, Biology Instructor 1 opyright The Mcraw Hill ompanies Inc. Permission required for reproduction or display

2 Outline enetic Material Transformation DNA Structure Watson and rick DNA Replication Prokaryotic versus Eukaryotic Replication Errors Transcription Translation Structure of Eukaryotic hromosome 2

3 enetic Material Frederick riffith investigated virulence of Streptococcus pneumoniae oncluded that virulence passed from the dead strain to the living strain Transformation Further research by Avery et al. Discovered that DNA is the transforming substance DNA from dead cells was being incorporated into genome of living cells 3

4 riffith 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. 4

5 riffith s Transformation Experiment opyright The Mcraw-Hill ompanies, Inc. Permission required for reproduction or display. capsule Injected live S strain has capsule and causes mice to die. Injected live R strain has no capsule and mice do not die. Injected heatkilled S strain does not cause mice to die. Injected heat-killed S strain plus live R strain causes mice to die. Live S strain is withdrawn from dead mice. a. b. c. d. 5

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7 Transformation of Organisms Today Result the so-called genetically modified organisms (MOs) Invaluable tool in modern biotechnology today ommercial products that are currently much used reen fluorescent protein (FP) used as a marker A jellyfish gene codes for FP 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 7

8 Transformation of Organisms opyright The Mcraw-Hill ompanies, Inc. Permission required for reproduction or display. A normal canola plant (left) and a transgenic canola plant expressing FP (right) under a fluorescent light. (Bacteria): Martin Shields/Photo Researchers, Inc.; (Jellyfish): R. Jackman/OSF/Animals Animals/Earth Scenes; (Pigs): ourtesy Norrie Russell, The Roslin Institute; (Mouse): Eye of Science/Photo Researchers, Inc.; (Plant): Dr. Neal Stewart 8

9 Structure of DNA DNA contains: Two Nucleotides with purine bases Adenine (A) uanine () Two Nucleotides with pyrimidine bases Thymine (T) ytosine () 9

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11 hargaff s Rules The amounts of A, T,, and in DNA: Identical in identical twins Varies between individuals of a species Varies more from species to species In each species, there are equal amounts of: A & T & All this suggests DNA uses complementary base pairing to store genetic info Human chromosome estimated to contain, on average, 140 million base pairs Number of possible nucleotide sequences, 4,140,000,000 11

12 Nucleotide omposition of DNA opyright The Mcraw-Hill ompanies, Inc. Permission required for reproduction or display. adenine (A) O N H 5 HO P O H 2 O 4 H H 3 NH 2 N OH O N N H 1 H 2 H H phosphate nitrogen-containing base guanine () O HO P H 2 N HN 5 O H 2 O N N N H HO 5 P O H 2 O 4 H H 1 H 3 H 2 a. Purine nucleotides OH H b. Pyrimidine nucleotides O thymine (T) O O 4 H H 3 O OH HN O N H 1 H 2 H H H 3 sugar = deoxyribose HO cytosine () O 5 P O H 2 O O O N NH 2 N 4 H H 1 H 3 H 2 OH H H H DN A omposition in V arious Species (%) Species A T Homo sapiens (human) Drosophila melanogaster (fruit fly) Zea mays (corn) Neurospora crassa (fungus) Escherichia coli (bacterium) Bacillus subtilis (bacterium) c. hargaf f s data 12

13 Animation Please note that due to differing operating systems, some animations will not appear until the presentation is viewed in Presentation Mode (Slide Show view). You may see blank slides in the Normal or Slide Sorter views. All animations will appear after viewing in Presentation Mode and playing each animation. Most animations will require the latest version of the Flash Player, which is available at

14 Animation Please note that due to differing operating systems, some animations will not appear until the presentation is viewed in Presentation Mode (Slide Show view). You may see blank slides in the Normal or Slide Sorter views. All animations will appear after viewing in Presentation Mode and playing each animation. Most animations will require the latest version of the Flash Player, which is available at 14

15 Watson and rick Model Watson and rick, 1953 onstructed a model of DNA Double-helix model is similar to a twisted ladder Sugar-phosphate backbones make up the sides Hydrogen-bonded bases make up the rungs Received a Nobel Prize in

16 Watson/rick Model of DNA opyright The Mcraw-Hill ompanies, Inc. Permission required for reproduction or display nm 3.4 nm 2 nm b. d. a. P 5 end 5 4 S c. 1 3 end S 5 P A T T A P sugar-phosphate backbone P P complementary base P sugar hydrogen bonds a: Kenneth Eward/Photo Researchers, Inc.; d: A. Barrington Brown/Photo Researchers, Inc. 16

17 X-Ray Diffraction of DNA 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 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. 17

18 X-Ray Diffraction of DNA opyright The Mcraw-Hill ompanies, Inc. Permission required for reproduction or display. Rosalind Franklin a. X-ray beam diffracted X-rays diffraction pattern rystalline DNA b. Photo Researchers, Inc.; c: Science Source/Photo Researchers, Inc. c. 18

19 Replication of DNA DNA replication is the process of copying a DNA molecule. Replication is semiconservative, with each strand of the original double helix (parental molecule) serving as a template (mold or model) for a new strand in a daughter molecule. 19

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21 Animation Please note that due to differing operating systems, some animations will not appear until the presentation is viewed in Presentation Mode (Slide Show view). You may see blank slides in the Normal or Slide Sorter views. All animations will appear after viewing in Presentation Mode and playing each animation. Most animations will require the latest version of the Flash Player, which is available at 21

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23 Replication: Eukaryotic DNA replication begins at numerous points along linear chromosome DNA unwinds and unzips into two strands Each old strand of DNA serves as a template for a new strand omplementary base-pairing forms new strand on each old strand Requires enzyme DNA polymerase 23

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26 Replication: Eukaryotic Replication bubbles spread bi-directionally until they meet The complementary nucleotides join 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. 26

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28 Semiconservative Replication of DNA opyright The Mcraw-Hill ompanies, Inc. Permission required for reproduction or display. 5' 3' A A T region of parental DNA double helix T A A T A A region of replication: new nucleotides are pairing with those of parental strands old strand T A A A A A new strand daughter DNA double helix T T T T A A region of completed replication T T A A A A A T T T new strand A A old strand daughter DNA double helix 28

29 Animation Please note that due to differing operating systems, some animations will not appear until the presentation is viewed in Presentation Mode (Slide Show view). You may see blank slides in the Normal or Slide Sorter views. All animations will appear after viewing in Presentation Mode and playing each animation. Most animations will require the latest version of the Flash Player, which is available at 29

30 Animation Please note that due to differing operating systems, some animations will not appear until the presentation is viewed in Presentation Mode (Slide Show view). You may see blank slides in the Normal or Slide Sorter views. All animations will appear after viewing in Presentation Mode and playing each animation. Most animations will require the latest version of the Flash Player, which is available at

31 Science Focus: Aspects of DNA Replication opyright The Mcraw-Hill ompanies, Inc. Permission required for reproduction or display. OH P is attached here 5 H 2 base is attached here O OH 4 H H 1 1 H H 3 2 OH H Deoxyribose molecule 5 end P 2 DNA polymerase attaches a new nucleotide to the 3 carbon of the previous nucleotide. P P P 3 end P P 5 P T A P 3 P P template strand 3 end template strand Direction of replication 5 lagging strand 5 template strand 5 end new strand 6 P 4 Okazaki fragment leading new strand 3 RNA primer DN A polymerase 3 helicase at replication fork parental DN A helix DNA ligase 3 Replication fork introduces complications DNA polymerase 31

32 Replication: Prokaryotic Prokaryotic Replication Bacteria have a single circular loop Replication moves around the circular DNA molecule in both directions Produces two identical circles ell divides between circles, as fast as every 20 minutes 32

33 Replication: Prokaryotic vs. Eukaryotic opyright The Mcraw-Hill ompanies, Inc. Permission required for reproduction or display. origin replication is complete replication is occurring in two directions a. Replication in prokaryotes replication fork replication bubble parental strand new DNA duplexes b. Replication in eukaryotes daughter strand 33

34 Replication Errors enetic variations are the raw material for evolutionary change Mutation: A permanent (but unplanned) change in basepair sequence Some due to errors in DNA replication Others are due to to DNA damage DNA repair enzymes are usually available to reverse most errors 34

35 Function of enes enes Specify Enzymes Beadle and Tatum: Experiments on fungus Neurospora crassa Proposed that each gene specifies the synthesis of one enzyme One-gene-one-enzyme hypothesis enes Specify a Polypeptide A gene is a segment of DNA that specifies the sequence of amino acids in a polypeptide Suggests that genetic mutations cause changes in the primary structure of a protein 35

36 Protein Synthesis: From DNA to RNA to Protein The mechanism of gene expression DNA in genes specify information, but information is not structure and function enetic info is expressed into structure & function through protein synthesis The expression of genetic info into structure & function: DNA in gene controls the sequence of nucleotides in an RNA molecule RNA controls the primary structure of a protein 36

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38 The entral Dogma of Molecular Biology opyright The Mcraw-Hill ompanies, Inc. Permission required for reproduction or display. nontemplate strand 5' 3' A A DNA T T 3' 5' transcription in nucleus template strand 5' 3' mrn A A translation at ribosome codon 1 codon 2 codon 3 O O O N N N R 1 R 2 R 3 polypeptide Serine Aspartate Proline 38

39 Types of RNA RNA is a polymer of RNA nucleotides RNA Nucleotides are of four types: Uracil, Adenine, ytosine, and uanine Uracil (U) replaces thymine (T) of DNA Types of RNA Messenger (mrna) - Takes genetic message from DNA in nucleus to ribosomes in cytoplasm Ribosomal (rrna) - Makes up ribosomes which read the message in mrna Transfer (trna) - Transfers appropriate amino acid to ribosome when instructed 39

40 Structure of RNA opyright The Mcraw-Hill ompanies, Inc. Permission required for reproduction or display. P U S A P S U base is uracil instead of thymine P A S P S ribose one nucleotide 40

41 RNA vs. DNA structure 41

42 The enetic ode 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 ontains 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 42

43 The enetic ode The unit of a 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 enetic alphabet has only four letters (U, A,, ) odons 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 enetic language only has 64 words 43

44 Messenger RNA odons opyright The Mcraw-Hill ompanies, Inc. Permission required for reproduction or display. First Base Second Base U A UUU phenylalanine UU serine UAU tyrosine UU cysteine Third Base U U UU phenylalanine U serine UA tyrosine UU cysteine UUA leucine UA serine UAA stop UA stop A UU leucine U serine UA stop U tryptophan UU leucine U proline AU histidine U arginine U U leucine UA leucine proline A proline A histidine AA glutamine arginine A arginine A U leucine proline A glutamine arginine AUU isoleucine AU threonine AAU asparagine AU serine U A AU isoleucine AUA isoleucine A threonine AA threonine AA asparagine AAA lysine A serine AA arginine A AU (start) methionine A threonine AA lysine A arginine UU valine U alanine AU aspartate U glycine U U valine UA valine alanine A alanine A aspartate AA glutamate glycine A glycine A U valine alanine A glutamate glycine 44

45 Steps in ene Expression: Transcription Transcription ene unzips and exposes unpaired bases Serves as template for mrna formation Loose RNA nucleotides bind to exposed DNA bases using the = & A=U rule When entire gene is transcribed into mrna, result is a pre-mrna transcript of the gene The base sequence in the pre-mrna is complementary to the base sequence in DNA 45

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47 Transcription of mrna A single chromosomes 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 47

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49 Transcription opyright The Mcraw-Hill ompanies, Inc. Permission required for reproduction or display. 5' T A A T nontemplate strand template strand 3' A A RNA polymerase DNA template strand mrna transcript A T T A 5' 3' to RNA processing 49

50 Animation Please note that due to differing operating systems, some animations will not appear until the presentation is viewed in Presentation Mode (Slide Show view). You may see blank slides in the Normal or Slide Sorter views. All animations will appear after viewing in Presentation Mode and playing each animation. Most animations will require the latest version of the Flash Player, which is available at

51 Animation Please note that due to differing operating systems, some animations will not appear until the presentation is viewed in Presentation Mode (Slide Show view). You may see blank slides in the Normal or Slide Sorter views. All animations will appear after viewing in Presentation Mode and playing each animation. Most animations will require the latest version of the Flash Player, which is available at 51

52 RNA Polymerase opyright The Mcraw-Hill ompanies, Inc. Permission required for reproduction or display. a. 200m spliceosome DNA RNA polymerase RNA transcripts b. Oscar L. Miller/Photo Researchers, Inc. 52

53 Processing Messenger RNA Pre-mRNA, is modified before leaving the eukaryotic nucleus. Modifications to ends of primary transcript: ap of modified guanine on 5 end The cap is a modified guanine () nucleotide Helps a ribosome where to attach when translation begins Poly-A tail of 150+ adenines on 3 end Facilitates the transport of mrna out of the nucleus Inhibits degradation of mrna by hydrolytic enzymes. 53

54 Processing Messenger RNA 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 mature mrna transcript 54

55 RNA Splicing 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 55

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57 Messenger RNA Processing opyright The Mcraw-Hill ompanies, Inc. Permission required for reproduction or display. DNA exon exon exon intron intron transcription exon exon exon pre-mrna 5' intron intron 3' exon exon exon 5' 3' cap intron intron poly-a tail spliceosome exon exon exon 5' 3' cap pre-mrna splicing poly-a tail intron RNA mrna 5' 3' cap poly-a tail nucleus nuclear pore in nuclear envelope cytoplasm 57

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59 Functions of Introns As organismal complexity increases; Number of protein-coding genes does not keep pace But the proportion of the genome that is introns increases Humans: enome has only about 25,000 coding genes Up to 95% of this DNA genes is introns Possible functions of introns: More bang for buck Exons might combine in various combinations Would allow different mrnas to result from one segment of DNA Introns might regulate gene expression Exciting new picture of the genome is emerging 59

60 Steps in ene Expression: Translation 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 species of trna Translation An mrna transcript migrates to rough endoplasmic reticulum Associates with the rrna of a ribosome The ribosome reads the information in the transcript Ribosome directs various species of trna to bring in their specific amino acid fares trna specified is determined by the code being translated in the mrna transcript 60

61 trna trna molecules come in 64 different kinds All very similar except that One end bears a specific triplet (of the 64 possible) called the anticodon Other end binds with a specific amino acid type trna synthetases attach correct amino acid to the correct trna molecule All trna molecules with a specific anticodon will always bind with the same amino acid 61

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63 Structure of trna opyright The Mcraw-Hill ompanies, Inc. Permission required for reproduction or display. amino acid leucine 3 5 Hydrogen bonding amino acid end anticodon anticodon end mrna 5' codon 3' b. 63

64 Ribosomes Ribosomal RNA (rrna): Produced from a DNA template in the nucleolus ombined 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 64

65 Ribosomal Structure and Function opyright The Mcraw-Hill ompanies, Inc. Permission required for reproduction or display. large subunit 5 3 mrna trna binding sites a. Structure of a ribosome small subunit b. Binding sites of ribosome outgoing trna polypeptide incoming trna incoming trna c. Function of ribosomes d. Polyribosome ourtesy Alexander Rich 65

66 Steps in Translation: Initiation omponents 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 UA anticodon Always carries the amino acid methionine apable of binding to the P site 66

67 Steps in Translation: Initiation Small ribosomal subunit attaches to mrna transcript Beginning of transcript always has the START codon (AU) Initiator trna (UA) attaches to P site Large ribosomal subunit joins the small subunit 67

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69 Steps in Translation: Initiation opyright The Mcraw-Hill ompanies, Inc. Permission required for reproduction or display. amino acid methionine Met 5' mrna initiator trna E site P site A site 3' Met small ribosomal subunit large ribosomal subunit U A A U 5' 3' start codon A small ribosomal subunit binds to mrna; an initiator trna pairs with the mrna start codon AU. The large ribosomal subunit completes the ribosome. Initiator trna occupies the P site. The A site is ready for the next trna. Initiation 69

70 Steps in Translation: 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 Joins the ribosome at it s A site Methionine of initiator is connected to amino acid of 2 nd trna by peptide bond 70

71 Steps in Translation: Elongation Second trna moves to P site (translocation) Spent initiator moves to E site and exits 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 it s A site Dipeptide on 2 nd amino acid is connected to amino acid of 3 nd trna by peptide bond 71

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73 Steps in Translation: Elongation opyright The Mcraw-Hill ompanies, Inc. Permission required for reproduction or display. Met Ser Ala peptide bond Trp Val asp U Met trna Ser Ala anticodon Trp Val Asp Met Ser Ala Trp Val Asp peptide bond Met Ser Ala Trp Val Asp Thr A U U A A A U U A U A A U U A U A U A U A A A trna amino acid approaches the ribosome and binds at the A site. 2 Two trnas can be at a ribosome at one time; the anticodons are paired to the codons. 3 Peptide bond formation attaches the peptide chain to the newly arrived amino acid. 4 The ribosome moves forward; the empty trna exits from the E site; the next amino acid trna complex is approaching the ribosome. Elongation 73

74 Steps in Translation: Termination Previous trna moves to P site Spent trna moves to E site and exits Ribosome reads the STOP codon at the end of the mrna UAA, UA, or UA Does not code for an amino acid Polypeptide is released from last trna by release factor Ribosome releases mrna and dissociates into subunits mrna read by another ribosome 74

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76 Steps in Translation: Termination opyright The Mcraw-Hill ompanies, Inc. Permission required for reproduction or display. Asp Ala Trp Val lu release factor U U A A U A 5' stop codon 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 76

77 Animation Please note that due to differing operating systems, some animations will not appear until the presentation is viewed in Presentation Mode (Slide Show view). You may see blank slides in the Normal or Slide Sorter views. All animations will appear after viewing in Presentation Mode and playing each animation. Most animations will require the latest version of the Flash Player, which is available at 77

78 Summary of ene Expression (Eukaryotes) opyright The Mcraw-Hill ompanies, Inc. Permission required for reproduction or display. TRANSRIPTION DNA pre-mrna introns mrna 1. DNA in nucleus serves as a template for mrna. 2. mrna is processed before leaving the nucleus. 3' nuclear pore ribosome mrna TRANSLATION peptide large and small ribosomal subunits amino acids 5 trna 3. mrna moves into cytoplasm and becomes associated with ribosomes. 4. trnas with anticodons carry amino acids to mrna. 5 U A A U 3 anticodon codon 5 U U A A U A U A 5. During initiation, anticodon-codon complementary base pairing begins as the ribosomal subunits come together at a start codon. 3' 8. During termination, a ribosome reaches a stop codon; mrna and ribosomal subunits disband. 6. During elongation, polypeptide synthesis takes place one amino acid at a time. 7. Ribosome attaches to rough ER. Polypeptide enters lumen, where it folds and is modified. 78

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80 Structure of Eukaryotic hromosome ontains 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 Five primary types of histone molecules Responsible for packaging the DNA DNA double helix is wound at intervals around a core of eight histone molecules (called nucleosome) Nucleosomes are joined by linker DNA. 80

81 Structure of Eukaryotic hromosome opyright The Mcraw-Hill ompanies, Inc. Permission required for reproduction or display. 2 nm a. Nucleosomes ( beads on a string ) 1 nm histones DNA double helix 1. Wrapping of DNA around histone proteins. nucleosome b. 30-nm fiber 30 nm histone H1 2. Formation of a three-dimensional zigzag structure via histone H1 and other DNA-binding proteins. 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 81

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83 Review enetic Material Transformation DNA Structure Watson and rick DNA Replication Prokaryotic versus Eukaryotic Replication Errors Transcription Translation Structure of Eukaryotic hromosome 83

84 hapter 12: pp BIOLOY 10th Edition Molecular Biology of the ene 2 nm opyright The Mcraw-Hill ompanies, Inc. Permission required for reproduction or display. b nm 3.4 nm T sugar-phosphate backbone Sylvia S. Mader P A T A P P P Sugar hydrogen bonds PowerPoint Lecture Slides are prepared by Dr. Isaac Barjis, Biology Instructor 84 opyright The Mcraw Hill ompanies Inc. Permission required for reproduction or display