Chapter 12 Molecular Biology of the Gene PDF

Because learning changes everything. ® Biology Sylvia S. Mader...

Because learning changes everything. ® Biology Sylvia S. Mader Michael Windelspecht Chapter 12 Molecular biology of the Gene Copyright 2022 © McGraw Hill LLC. All rights reserved. No reproduction or distribution without the prior written consent of McGraw Hill LLC.  2 Outline  12.1 The Genetic Material  12.2 Replication of DNA  12.3 Gene Expression: RNA & The Genetic Code  12.4 Gene Expression: Transcription  12.5 Gene Expression: Translation  12.1 The Genetic Material Scientists knew that genetic material must be able to: 1. Store information related to development, structure & metabolic activity of the cell. 2. Stable so it can be replicated with accuracy. & can be transmitted from generation to generation. 3. Able to undergo mutations to provide genetic variability. In the nucleus, nucleic acids were discovered: DNA: deoxyribonucleic acid RNA: ribonucleic acid  Transformation of Bacteria  1920, F. Griffith was trying to develop a vaccine for Streptococcus pneumoniae. - S strain bacteria (shiny, smooth, encapsulated, virulent) - R strain bacteria (rough, nonencapsulated, nonvirulent) Is the capsule alone responsible for the virulence? - Heat killed S strain  mice don’t die - Heat killed S strain + live R strain mice die + living S strain strain was recovered.  Griffith’s Conclusion - Some substance necessary for the bacteria to produce a capsule & be virulent must have passed from killed S strain to R strain so that the R strain were transformed. - There is a substance responsible for the transformation. - A change in the phenotype of the R strain must be due to a change in the genotype.  Is it the DNA or the Protein ?  Chromatin material contain DNA & proteins.  DNA & proteins were the candidates for the hereditary material. Proteins contain 20 amino acids that can be sequenced in different ways. DNA and RNA each contain only four types of nucleotides.  What Is The Genetic Material?  The chromosomes contain both DNA & proteins.  Nucleic acids: contain only 4 types of nucleotides (nitrogenous base + phosphate group + pentose).  Proteins : have 20 AA. It was thought that protein was the genetic material, due to greater room for variation.  DNA: The Transforming Substance 1940, Avery demonstrated that the transforming substance is DNA. 1. DNA from S strain caused R strain to transform, produce a capsule & become virulent. 2. Addition of enzymes that degrade proteins or RNA can’t prevent transformation. 3. Addition of DNAases, enzymes that degrade DNA, prevents transformation. 4. Mol. Wt. of transforming substance is large, so possibility of genetic variability.  Hershey & Chase confirmation Bacteriophages: - T phage= viruses that infect E. coli bacteria. - consist only of a protein capsid surrounding a nucleic acid core. - they discovered that the radioactively labeled DNA is the one that enters the bacteria, causing them to become transformed.  Reproduction of Viruses  Phosphorus P is present in DNA but not in protein.  Sulfur S is present in protein but not in DNA.  They used radioactive 32P and 35S as labels to detect where each of the substances was going. Hershey-Chase Experiment  Results indicate that the DNA, not the protein, enters the host.  DNA is the genetic material.  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 http://get.adobe.com/flashplayer.  The Structure of DNA DNA has 4 different types of nucleotides:  2 with purine (double ringed) bases: - Adenine (A) - Guanine (G)  2 with pyrimidine (single ringed) base - Thymine (T) - Cytosine (C)  The Structure of DNA  Chargaff’s rules: 1. The amount of A, T, G, and C in DNA varies from species to species. 1. In each species, A=T G=C.  A is always paired with T, and G is always paired with C.  Each human chromosome contains about 140 M base pairs.  The Structure of DNA  Franklin studied the structure of DNA using X-ray diffraction DNA has a helical shape with repeated portions.  Watson and Crick: DNA is a double helix, sugar- phosphate backbones on outside, paired bases on the inside.  The Structure of DNA  Watson and Crick: - The spacing between base pairs is 0.34 nm and for a complete turn of the double helix 3.4 nm. - A pairs with T and G pairs with C (complementary base pairing). - The two strands of the molecule are antiparallel. The 5’ end of one strand is opposite to the 3’ end of the other strand. Watson and Crick Model of DNA 3.4 nm 0.34 nm 17 2 nm b. d. C a. sugar-phosphate G T backbone 5′ end 3′ end P A P G C S S A T P P P T A S G S P C 3′ end 5′ end P complementary C c. base pairing G P hydrogen sugar bonds  Animation 18 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 http://get.adobe.com/flashplayer.  12.2 Replication of DNA  DNA replication: process of copying a DNA molecule.  It is semiconservative: each old DNA strand serves as a template for the new strand, and one old strand is in each new molecule.  Replication is Semiconservative  In 1958, Meselson and Stahl grew bacteria in 15N medium. They then transferred it to 14 N.  After one division: hybrid DNA molecules.  After two divisions: half light, half hybrid.  Expected results of semiconservative replication.  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 http://get.adobe.com/flashplayer.  Steps of DNA Replication 1.Unwinding: helicase unwinds at origin of replication (break H-bonds). Single stranded binding proteins (SSB) stabilize opened strands. 2. Complementary base pairing: Primase adds an RNA primer on which the DNA polymerase position the complementary nucleotides. DNA polymerase has a proof reading ability. 3. Joining: Ligase join okazaki fragments. complementary nucleotides form new strands. Information in DNA is always read In 3’ to 5’ direction  Semiconservative Replication 5′ 3′ 23 G C G C G A T A region of parental T A DNA double helix C G DNA A T polymerase A enzyme T G G T C G C G C G C A A C A T C G C region of G T A replication: T T A new nucleotides A T are pairing T with those of A G parental strands C T C A A G T A T A T A A G A G T G T region of A completed C C C A G replication C G 3′ new old A strand strand 5′ daughter DNA double helix old new strand strand daughter DNA double helix  OH P Is attached here 24 base is attached here 5′ CH2 O OH 4′ C C H H 1′ 1 H H 3′ C C 2′ OH H De ox yrib os e m o lec u le 2 DNA polymerase 5′ end attaches a new nucleotide to the P 3 ′ carbon of the previous nucleotide. T P A P G C 3′ e nd P P P C G 5′ P 3′ P T A C P template P G strand P DNA polymerase 3 ′end 5′ end 4 leading template strand new strand new strand Direction of replication 3′ 3 helicase at replication fork RNA primer template 6 Okazaki fragment lagging strand 5 strand 3′ 5′ 5′ parental DNA helix 7 DNA ligase DNA polymerase 3′ Replication fork introduces complications  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 http://get.adobe.com/flashplayer.  Prokaryotic Replication Bacteria Single circular loop of DNA. Replication occurs in two directions at once. The new strand is always in the 5’-3’ direction. In prokaryotes there is only one origin of replication.  Eukaryotic Replication. Replication begins at numerous origins of replicationreplication bubbles. In each we have 2 replication forks. Telomeres , repeated DNA sequences at ends of chromosomes that do not code for proteins. They are not copied by DNA polymerase but by Telomerases. DNA gets shorter after each replication.  Prokaryotic versus Eukaryotic Replication origin 28 replication is complete replication is occurring in two directions a. Replication in prokaryotes replication fork replication bubble parental strand new DNA duplexes daughter strand b. Replication in eukaryotes  Accuracy of Replication  DNA polymerase is very accurate. It makes one mistake/ 100,000 base pairs.  Genetic mutation: permanent change in the sequence of bases.  Some are due to errors in DNA replication. Polymerases have proofreading activity.  Some are due to DNA damage. DNA repair enzymes may correct many of these errors.  Errors don’t necessarily harm the organism. They are the material for evolutionary process.  12.3 Gene Expression: RNA & the Genetic code  1940, Beadle and Tatum performed experiments on Neurospora ( red bread mold) and proposed the “one gene-one enzyme hypothesis”.  A defective gene caused a defective enzyme.  Genes Specify a Polypeptide  Pauling demonstrated from sickle cell anemia that a mutation leads to a change in the structure of a protein. In sickle cell anemia only the β chain in hemoglobin is affected. one gene-one enzyme hypothesis became one gene-one polypeptide hypothesis. The Central Dogma of Molecular Biology nontemplate strand 5 3 A G C G A C C C C DNA T C G C T G G G G 3 5 template strand transcription in nucleus 5 3 A G C G A C C C C mRN A translation codon 1 codon 2 codon 3 at ribosome O O O polypeptide N C C N C C N C C R1 R2 R3 32 Serine Aspartate Proline  RNA carries the information -A polymer of nucleotides. - has a ribose sugar (not deoxyribose) - has 4 different bases: A,C,G,U - 3 types of RNA 1. Messenger (mRNA) - Takes a message from DNA in the nucleus to ribosomes in the cytoplasm. 2. Ribosomal (rRNA) – with proteins make up ribosomes, which read the message in mRNA. 3. Transfer (tRNA) - Transfers the appropriate amino acid to the ribosomes for protein synthesis  Gene Expression Gene expression involves two steps: 1. Transcription from DNA to mRNA 2. Translation from mRNA to protein  Transcription  in the nucleus  leads to mRNA.  RNA polymerases attaches to a promoter that initiates transcription.  mRNA has a complementary sequence to the DNA template.  DNA helix unwinds complementary RNA nucleotides pair with DNA nucleotides  RNA polymerase reads the DNA template in the 3’5’ direction.  Transcription  Begins when RNA polymerase attaches to promoter.  Promoter defines start of a gene, direction of transcription, & strand to be transcribed.  DNA-RNA association is not stable.  Terminator causes RNA polymerase to stop and to release mRNA  RNA transcript.  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 http://get.adobe.com/flashplayer.  Messenger RNA is processed - Primary mRNA transcript in eukaryotes get modified before leaving the nucleus. - Modifications  cap on 5’ end (modified G)  tells the ribosome where to attach during translation.  poly-A tail on 3’ end  Facilitates transport of mRNA out of nucleus.  Inhibits degradation of mRNA by hydrolytic enzymes.  Helps loading of ribosome.  RNA Splicing Performed by spliceosomes which contain snRNAs that identify the introns & uses Ribozymes to remove introns.  Introns, are excised (removed)  Exons (protein coding) are spliced back together.  Result a mature mRNA transcript. Not all exons are translated !!!  Animation Functions of introns: As organismal complexity increases; The number of protein-coding genes does not keep pace PleaseBut the note thatproportion of the due to differing genome operating systems,that someis introns animations increases will not appear until the presentation is Possible functions ofMode viewed in Presentation introns: (Slide ShowExons view). Youmight combine may see in blank slides in the “Normal” or “Slide Sorter” views. various combinations All animations will appear after viewing Would allow different mRNAs in Presentation Mode and playing each animation.to result Most from one animations will segment require of DNA of the Flash Player, the latest version whichIntrons might is available at regulate gene expression http://get.adobe.com/flashplayer. Introns may encourage crossing-over during meiosis Exciting new picture of the genome is emerging 41 Possible functions of introns:  Alternative mRNA splicing: Introns allows the cell to pick and choose which exons will go into a particular mRNA. Different mRNAs will result from one segment of DNA.  Introns give rise to microRNAs that regulate gene expression since they can bind to mRNA bases and prevent translation.  Exon shuffling: Introns may encourage crossing-over during meiosis  The Genetic Code is universal - Triplet code - 3 nucleotides 1 codon 1 amino acid. - 64 possible arrangements of four symbols taken three at a time. -1961, Nirenberg & Matthei constructed a synthetic RNA (UUU) & were able to translate it in a test tube that contains cytoplasmic contents of the cell.  The Genetic Code Properties of the genetic Code: 1. Degenerate: most amino acids have more than one codon  protects against harmful mutations. 2. Unambiguous: Each triplet codon has only one meaning. 3. Code has one start & 3 stop signals. Universal  Translation  Takes place in cytoplasm of eukaryotic cells.  tRNA transfer amino acids to the ribosomes.  tRNA is folded on itself, has cloverleaf structure.  Amino acid binds to 3’ end of tRNA by an enzyme aminoacyl-tRNA synthtases. : activating enzymes responsible for attaching the correct amino acid to tRNA. An energy requiring process.  Translation  45 different tRNA molecules Each of the 20 amino acids in proteins associates with one or more of 45 types of tRNA.  The “wobble” hypothesis predicts that the third position in the tRNA anticodon doesn’t obey the A-U/G-C configuration rule and can be variable  The Role of Ribosomal RNA  rRNA is produced in the nucleolus of the nucleus.  Packaged with proteins into 2 separate ribosomal subunits.  In the cytoplasm they combine on the mRNA and can initiate translation.  Ribosomes have a 3 binding site E, P, A.  large rRNA subunit has an enzyme activity that creates a peptide bond between adjacent amino acids.  The Role of Ribosomal RNA  Translation terminates when polypeptide is fully formed ribosome dissociates into 2 subunits.  Several ribosomes are attached the same mRNA =>Polyribosome.  The folding process begins when polypeptide emerges from ribosome.  Chaperons ensure proper folding of the protein.. Three steps of Translation: 1-Initiation 2- Elongation 3- Termination  Initiation 49 amino acid methionine Met initiator tRNA UA 5 AU C mRNA E site P site A site G 3 Met small ribosomal subunit large ribosomal subunit U AC AUG 5 start codon 3 A small ribosomal subunit binds to mRNA; an initiator tRNA pairs with the mRNA start codon AUG. The large ribosomal subunit completes the ribosome. Initiator tRNA occupies the P site. The A site is ready for the next tRNA. Initiation Elongation Translocation occurs: mRNA with the peptide-bearing Met tRNA moves to the P site and the spent tRNA moves peptide from P to E site. Ser bond Ala Trp Val C A U G U A G A C 5 3 50 Elongation asp Met peptide tRNA Ser bond Ala C U G Trp anticodon Val C A U G U A G A C 3 5 1 A tRNA–amino acid approaches the ribosome and binds at the A site. 51  Elongation asp Met Met peptide tRNA Ser bond Ser Ala C UG Ala Trp anticodon Trp Val Val Asp C A U C A U C UG G UA GA C G UA GA C 3 3 5 5 1 2 A tRNA–amino acid Two tRNAs can be at a approaches the ribosome at one time; ribosome and binds the anticodons are at the A site. paired to the codons. 52  Elongation Met Met asp Met Met Ser Thr Ser peptide tRNA Ser bond Ser Ala Ala Ala C U G Ala Trp Trp peptide U G G Trp anticodon Trp Val bond Val Val Val Asp Asp Asp U A C C A U C A U C U G C A U C U G C U G G U A G A C G U A G A C A C C G U A G A C G U A G A C 3 5 3 5 5 3 5 3 1 A tRNA–amino acid 2 Two tRNAs can be at a 3 Peptide bond formation 4 The ribosome moves forward; the approaches the ribosome at one time; attaches the peptide “empty” tRNA exits from the E site; ribosome and binds the anticodons are chain to the newly the next amino acid–tRNA complex at the A site. paired to the codons. arrived amino acid. is approaching the ribosome. 53  Termination 54 Asp Ala Trp Asp release factor Val Ala Glu Trp Val U U A A U G A Glu 5′ stop codon 3′ U The ribosome comes to a stop U C codon on the mRNA. A release factor binds to the site. A A U G G A 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  Summary of Protein Synthesis in Eukaryotes TRANSCRIPTION 1. DNA in nucleus serves TRANSLATION as a template for mRNA. DNA 3. mRNA moves into 2. mRNA is processed large and small cytoplasm and before leaving the nucleus. ribosomal subunits 5 becomes associated mRNA with ribosomes. introns pre-mRNA 3 mRNA amino 4. tRNAs with acids nuclear pore anticodons carry amino acids peptide to mRNA. ribosome tRNA U A U A C C 5 A UG 3 anticodon codon 5. During initiation, anticodon-codon complementary base pairing begins CC 8. During termination, a C as the ribosomal subunits come together at a start codon. ribosome reaches a stop CCC UGG UU U codon; mRNA and 5 GGG ACC AA A GUA ribosomal subunits 3 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. 55  Protein Synthesis & the Eukaryotic Cell  Eukaryotes:  mRNA produced and processed in the nucleus  translation in the cytoplasm.  Some ribosomes free in cytoplasm.  Others attached to rough ER  Protein Synthesis & the Eukaryotic Cell  -1st few A.A of a polypeptide act as a signal to where the polypeptide belongs in the cell.  In the lumen of the ER polypeptide is folded and processed by addition of sugars, phosphates, or lipids.  -Transport vesicles carry proteins between organelles.  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 http://get.adobe.com/flashplayer. 58  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 http://get.adobe.com/flashplayer.