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Essentials of The Living World First Edition GEORGE B. JOHNSON 9 How Genes Work PowerPoint® Lectures prepared by Johnny El-Rady Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 9.1 The Griffith Experiment Mendel’s work left a key question unanswered: What is a gene? The work of Sutton and Morgan established that genes reside on chromosomes But chromosomes contain proteins and DNA So which one is the hereditary material Several experiments ultimately revealed the nature of the genetic material Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 9.1 The Griffith Experiment In 1928, Frederick Griffith discovered transformation while working on Streptococcus pneumoniae The bacterium exists in two strains S Forms smooth colonies in a culture dish Cells produce a polysaccharide coat and can cause disease R Forms rough colonies in a culture dish Cells do not produce a polysaccharide coat and are therefore harmless Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Fig. 9.1 How Griffith discovered transformation Thus, the dead S bacteria somehow “transformed” the live R bacteria into live S bacteria Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 9.2 The Avery and Hershey-Chase Experiments Two key experiments that demonstrated conclusively that DNA, and not protein, is the hereditary material Oswald Avery and his coworkers Colin MacLeod and Maclyn McCarty published their results in 1944 Alfred Hershey and Martha Chase published their results in 1952 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display The Avery Experiments Avery and his colleagues prepared the same mixture of dead S and live R bacteria as Griffith did They then subjected it to various experiments All of the experiments revealed that the properties of the transforming principle resembled those of DNA 1. 2. 3. 4. Same chemistry and physical properties as DNA Not affected by lipid and protein extraction Not destroyed by protein- or RNA-digesting enzymes Destroyed by DNA-digesting enzymes Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display The Hershey-Chase Experiment Viruses that infect bacteria have a simple structure DNA core surrounded by a protein coat Hershey and Chase used two different radioactive isotopes to label the protein and DNA Incubation of the labeled viruses with host bacteria revealed that only the DNA entered the cell Therefore, DNA is the genetic material Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Fig. 9.2 The Hershey-Chase Experiment Thus, viral DNA directs the production of new viruses Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 9.3 Discovering the Structure of DNA DNA is made up of nucleotides Each nucleotide has a central sugar, a phosphate group and an organic base The bases are of two main types Purines – Large bases Adenine (A) and Guanine (G) Pyrimidines – Small bases Cytosine (C) and Thymine (T) Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Fig. 9.3 The four nucleotide subunits that make up DNA Nitrogenous base 5-C sugar Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Erwin Chargaff made key DNA observations that became known as Chargaff’s rule Purines = Pyrimidines Rosalind Franklin’s X-ray diffraction experiments revealed that DNA had the shape of a coiled spring or helix A = T and C = G Fig. 9.4 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Rosalind Franklin (1920-1958) In 1953, James Watson and Francis Crick deduced that DNA was a double helix They came to their conclusion using Tinkertoy models and the research of Chargaff and Franklin Fig. 9.4 James Watson (1928) Francis Crick (1916-2004) Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Fig. 9.4 The DNA double helix Dimensions suggested by X-ray diffraction The two possible basepairs Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 9.4 How the DNA Molecule Replicates The two DNA strands are held together by weak hydrogen bonds between complementary base pairs A and T C and G If the sequence on one strand is The other’s sequence must be ATACGCAT TATGCGTA Each chain is a complementary mirror image of the other So either can be used as template to reconstruct the other Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display There are 3 possible methods for DNA replication Fig. 9.5 Daughter DNAs contain one old and one new strand Original DNA molecule is preserved Old and new DNA are dispersed in daughter molecules Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display These three mechanisms were tested in 1958 by Matthew Meselson and Franklin Stahl Fig. 9.6 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Thus, DNA replication is semi-conservative Fig. 9.6 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display How DNA Copies Itself The process of DNA replication can be summarized as such The enzyme helicase first unwinds the double helix The enzyme primase puts down a short piece of RNA termed the primer DNA polymerase reads along each naked single strand adding the complementary nucleotide Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Fig. 9.7 How nucleotides are added in DNA replication Template strand New strand HO 3’ HO 5’ C Sugarphosphate backbone Template strand P O P T A T P O A P O O P T A T A P P DNA polymerase O O O O P P C G C P O O G P O O P P A 3’ OH A O A T O O T P 5’ P G O P P 5’ C O O O O 3’ P G New strand P P P Pyrophosphate P P O A O OH P 5’ Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 3’ OH DNA polymerase can only build a strand of DNA in one direction The leading strand is made continuously from one primer The lagging strand is assembled in segments created from many primers Fig. 9.8 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display RNA primers are removed and replaced with DNA Ligase joins the ends of newly-synthesized DNA Fig. 9.9 Mechanisms exist for DNA proofreading and repair Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 9.5 Transcription The path of genetic information is often called the central dogma DNA RNA Protein A cell uses three kinds of RNA to make proteins Messenger RNA (mRNA) Transfer RNA (tRNA) Ribosomal RNA (rRNA) Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 9.5 Transcription Gene expression is the use of information in DNA to direct the production of proteins It occurs in two stages Fig. 9.10 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 9.5 Transcription The transcriber is RNA polymerase It binds to one DNA strand at a site called the promoter It then moves along the DNA pairing complementary nucleotides It disengages at a stop signal Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Fig. 9.11 9.6 Translation Translation converts the order of the nucleotides of a gene into the order of amino acids in a protein The rules that govern translation are called the genetic code mRNAs are the “blueprint” copies of nuclear genes mRNAs are “read” by a ribosome in threenucleotide units, termed codons Each three-nucleotide sequence codes for an amino acid or stop signal Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Fig. 9.12 The genetic code is (almost) universal Only a few exceptions have been found Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Ribosomes The protein-making factories of cells They use mRNA to direct the assembly of a protein A ribosome is made up of two subunits Each of which is composed of proteins and rRNA Fig. 9.13 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Sites play key roles in translation Transfer RNA tRNAs bring amino acids to the ribosome They have two business ends Anticodon which is complementary to the codon on mRNA 3’–OH end to which the amino acid attaches Hydrogen bonding causes hairpin loops 3-D shape Fig. 9.14 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Making the Protein mRNA binds to the small ribosomal subunit The large subunit joins the complex, forming the complete ribosome mRNA threads through the ribosome producing the polypeptide Fig. 9.16 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Fig. 9.15 How translation works The process continues until a stop codon enters the A site The ribosome complex falls apart and the protein is released Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 9.7 Architecture of the Gene In eukaryotes, genes are fragmented They are composed of Exons – Sequences that code for amino acids Introns – Sequences that don’t Eukaryotic cells transcribe the entire gene, producing a primary RNA transcript This transcript is then heavily processed to produce the mature mRNA transcript This leaves the nucleus for the cytoplasm Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Fig. 9.17 Processing eukaryotic mRNA Protect from degradation and facilitate translation Different combinations of exons can generate different polypeptides via alternative splicing Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 6. The polypeptide chain grows until the protetin is completed. 7. Phosphorylation or other chemical modifications can alter the activity of a protein after it is translated. Amino acid Completed polypeptide tRNA 5’ Ribosome moves toward 3’ end Cytoplasm Fig. 9.18 How protein synthesis works in eukaryotes Ribosome 5. tRNAs bring their amino acids in at the A site of the ribosome. Peptide bonds form between amino acids at the P site, and tRNAs exit the ribosome from the E site. 4. tRNA molecules become attached to specific amino acids with the help of activating enzymes. Amino acids are brought to the ribosome in the order dictated by the mRNA. DNA Nuclear membrane 3’ 3’ RNA polymerase 1. In the cell nucleus, RNA polymerase transcribes RNA from DNA 3’ Poly-A tail 5’ 5’ 5’ 3’ Primary RNA transcript Exons Nuclear pore 5’ Cap Large ribosomal subunit mRNA Poly-A tail Introns 3’ Cap Small ribosomal subunit mRNA 2. Introns are excised from the RNA transcript, and the remaining exons are spliced together, producing mRNA 3. mRNA is transported out of the nucleus. In the cytoplasm, ribosomal subunits bind to the mRNA Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 9.7 Architecture of the Gene Most eukaryotic genes exist in multiple copies Clusters of almost identical sequences called multigene families As few as three and as many as several hundred genes Transposable sequences or transposons are DNA sequences that can move about in the genome They are repeated thousands of times, scattered randomly about the chromosomes Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 9.8 Turning Genes Off and On Genes are typically controlled at the level of transcription In prokaryotes, proteins either block or allow the RNA polymerase access to the promoter Repressors block the promoter Activators make the promoter more accessible Most genes are turned off except when needed Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display The lac Operon An operon is a segment of DNA that contains a cluster of genes that are transcribed as a unit The lac operon contains Three structural genes Encode enzymes involved in lactose metabolism Two adjacent DNA elements Promoter Site where RNA polymerase binds Operator Site where the lac repressor binds Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display The lac Operon In the absence of lactose, the lac repressor binds to the operator RNA polymerase cannot access the promoter Therefore, the lac operon is shut down Fig. 9.19 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display The lac Operon In the presence of lactose, a metabolite of lactose called allolactose binds to the repressor This induces a change in the shape of the repressor which makes it fall off the operator RNA polymerase can now bind to the promoter Transcription of the lac operon is ON Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Fig. 9.19 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display The lac Operon What if the cell encounters lactose, and it already has glucose? The bacterial cell actually prefers glucose! The lac operon is also regulated by an activator The activator is a protein called CAP It binds to the CAP-binding site and gives the RNA polymerase more access to the promoter However, a “low glucose” signal molecule has to bind to CAP before CAP can bind to the DNA Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Fig. 9.20 Activators and repressors of the lac operon Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Enhancers DNA sequences that make the promoters of genes more accessible to many regulatory proteins at the same time Usually located far away from the gene they regulate Common in eukaryotes; rare in prokaryotes Fig. 9.21 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 9.9 Mutation The genetic material can be altered in two ways Recombination Change in the positioning of the genetic material Mutation Change in the content of the genetic material Bithorax mutant Fig. 9.22 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 9.9 Mutation Mutation and recombination provide the raw material for evolution Evolution can be viewed as the selection of particular combinations of alleles from a pool of alternatives The rate of evolution is ultimately limited by the rate at which these alternatives are generated Mutations in germ-line tissues can be inherited Mutations in somatic tissues are not inherited They can be passed from one cell to all its descendants Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Kinds of Mutation Mutations are caused in one of two ways Errors in DNA replication Mispairing of bases by DNA polymerase Mutagens Agents that damage DNA Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Kinds of Mutation The sequence of DNA can be altered in one of two main ways Point mutations Alteration of one or a few bases Base substitutions, insertion or deletion Frame-shift mutations Insertions or deletions that throw off the reading frame Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Fig. 9.23 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Kinds of Mutation The position of genes can be altered in one of two main ways Transposition Movement of genes from one part of the genome to another Occurs in both eukaryotes and prokaryotes Chromosomal rearrangements Changes in position and/or number of large segments of chromosomes in eukaryotes Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Mutation, Smoking and Lung Cancer Agents that cause cancer are called carcinogens These are typically mutagens The hypothesis that chemicals cause cancer was first advanced in the 18th century Many investigations since then have determined that chemicals can cause cancer in both animals and humans For example, tars and other chemicals in cigarette smoke can cause cancer of the lung Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display