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Chapter 14 DNA Replication Learning Objectives • Diagram the process of eukaryotic vs. prokaryotic DNA replication • Describe the semiconservative process of DNA replication • Diagram the structure of DNA (ie what are based like? How are they paired, where is the sugar backbone located and its general overall shape) • Name the 4 enzymes involved in DNA synthesis and their functions • Assess the importance of telomeres and telomerase • Describe the process and importance of DNA proofreading during replication • List the function and components of histones DNA • Stores information in a double helix • Structure was postulated by Watson and Crick, based on Xray crystallography done by Rosalind Franklin • DNA molecule consists of two polynucleotide chains twisted around each other into a righthanded double helix • Each nucleotide of the chains consists of – Deoxyribose – A phosphate group – A base (adenine, thymine, guanine, or cytosine) Structure • Deoxyribose sugars are linked by phosphate groups to form a sugar– phosphate backbone • Two strands are held together by base pairs – Adenine–Thymine, Guanine–Cytosine • Each full turn of double helix is 10 base pairs 5' end Phosphate Deoxyribose (a 5-carbon sugar) Adenine (A) Guanine (G) Purines (double-ring structures) Thymine (T) Cytosine (C) Pyrimidines (single-ring structures) Hydroxyl group 3' end Fig. 14-4, p. 281 2 nm 5' end 3' end Distance between each pair of bases = 0.34 nm Each full twist of the DNA double helix = 3.4 nm 5-carbon sugar deoxyribose) Nitrogenous base (guanine) Phosphate group 3' end Hydrogen bond 5' end Fig. 14-6, p. 283 DNA replication • DNA polymerases are the primary enzymes of DNA replication • Helicases unwind DNA to expose template strands for new DNA synthesis • RNA primers provide the starting point for DNA polymerase to begin synthesizing a new DNA chain • One new DNA strand is synthesized continuously; the other, discontinuously Assembling Antiparallel Strands • Meselson and Stahl showed that DNA replication is semiconservative – Two strands of parental DNA molecule unwind – Each is a template for the synthesis of a complementary copy a. Semiconservative replication KEY Parental DNA Replicated DNA 1st replication 2nd replication The two parental strands of DNA unwind, and each is a template for synthesis of a new strand. After replication has occurred, each double helix has one old strand paired with one new strand. Fig. 14-8a, p. 285 Enzymes of DNA Replication • Helicase unwinds the DNA • Primase synthesizes RNA primer (starting point for nucleotide assembly by DNA polymerases) • DNA polymerases assemble nucleotides into a chain, remove primers, and fill resulting gaps • DNA ligase closes remaining single-chain nicks Telomerase • Ends of eukaryotic chromosomes • Short sequences repeated hundreds to thousands of times • Repeats protect against chromosome shortening during replication • Chromosome shortening is prevented in some cell types which have a telomerase enzyme (adds telomere repeats to chromosome ends) 3' end of template strand 1 3' end of DNA template unwound and ready for replication. 2 Primer added and new DNA assembled from end of primer. Primer New DNA 3 Primer removed. Gap left by primer removal Chromosome strand shortened Fig. 14-13, p. 291 Original end of chromosome 1 Extra telomere repeats added by telomerase at 3’ end of template strand Added telomere repeats 2 Primer added and gap filled in Primer added to chromosome end Gap filled in 3 Primer removed; original length is restored Primer removed Chromosome strand not shortened Fig. 14-14, p. 291 DNA Synthesis • Begins at sites that act as replication origins • Proceeds from the origins as two replication forks moving in opposite directions Origin DNA double helix Replication forks Replication direction Fig. 14-15, p. 292 Origin Replication forks DNA double helix Fig. 14-19, p. 297 Proofreading • If a replication error causes a base to be mispaired, DNA polymerase reverses and removes the most recently added base • Proofreading depends on the ability of DNA polymerases to reverse and remove mismatched bases • DNA repair corrects errors that escape proofreading Template strand DNA polymerase 1 Enzyme continues activity New strand in the forward direction as DNA 3’ polymerase as long as the most recently added nucleotide is correctly paired. 2 Enzyme adds a mispaired nucleotide. New strand 3 Enzyme reverses, acting as a deoxyribonuclease to remove the mispaired nucleotide. 4 Enzyme resumes forward activity as a DNA polymerase. Fig. 14-16, p. 293 Template strand Base-pair mismatch 1 Repair enzymes recognize a mispaired base and break one chain of the DNA at the arrows. New strand 2 The enzymes remove several to many bases, including the mismatched base, leaving a gap in the DNA. 3 The gap is filled in by a DNA polymerase using the intact template strand as a guide. Nick left after gap filled in 4 The nick left after gap filling is sealed by DNA ligase to complete the repair. Fig. 14-17, p. 293 DNA Organization in Eukaryotes and Prokaryotes • Histones pack eukaryotic DNA at successive levels of organization • Many nonhistone proteins have key roles in the regulation of gene expression • DNA is organized more simply in prokaryotes than in eukaryotes Chromatin • Distributed between: – Euchromatin (loosely packed region, genes active in RNA transcription) – Heterochromatin (densely packed masses, genes are inactive) • Folds and packs to form thick, rodlike chromosomes during nuclear division The Bacterial Chromosome • Closed, circular molecule of DNA packed into nucleoid region of cell • Replication begins from a single origin, proceeds in both directions • Plasmids (in many bacteria) replicate independently of the host chromosome Learning Objectives • Diagram the process of eukaryotic vs. prokaryotic DNA replication • Describe the semiconservative process of DNA replication • Diagram the structure of DNA (ie what are bases like? How are they paired, where is the sugar backbone located and its general overall shape) • Name the 4 enzymes involved in DNA synthesis and their functions • Assess the importance of telomeres and telomerase • Describe the process and importance of DNA proofreading during replication • List the function of histones Chapter 16: Gene regulation • Diagram the lac operon transcription unit • Compare and contrast the operon model of tryptophan and lactose metabolism • Compare and contrast prokaryotic and eukaryotic gene regulation Gene Expression Control All somatic cells in an organism are genetically identical – Cells differentiate by gene expression • Gene expression is collectively controlled through transcriptional regulation – Main control: Gene transcribed into mRNA – Additional controls: Posttranscriptional, translational and posttranslational Prokaryotic Gene Expression • Operon is the unit of transcription in prokaryotes • lac operon for lactose metabolism is transcribed when an inducer inactivates a repressor • Transcription of the lac operon is also controlled by a positive regulatory system Operon: Unit of Transcription • Prokaryotic gene expression reflects life history – Rapid, reversable response to environment • Operon: A cluster of prokaryotic genes and DNA sequences involved in their regulation – RNA polymerase binds at promoter for operon – Many genes may be transcribed into one mRNA – Cluster of genes is transscriptional unit Operon: Unit of Transcription (2) • Regulatory proteins bind at operator – Regulatory protein coded by gene outside operon • Repressor proteins prevent operon genes from being expressed • Activator proteins turn on expression of genes from operon lac Operon for Lactose Metabolism • Lactose metabolism in E. Coli requires three genes lacZ, lacY and lacA – lac operon contains all three genes and regulatory sequences • lac operon operator sequence is between promoter and lacZ Sequences that control the expression of the operon Regulatory gene lacI lac operon Promoter Operator lacZ lacY lacA DNA Binds RNA Binds Lac polymerase repressor Transcription initiation site Lac repressor β-Galactosidase Transcription termination site Permease Transacetylase Fig. 16-2, p. 331 lac Operon for Lactose Metabolism • lac repressor stops lac operon expression – Encoded by lacI, synthesized in active form – Binds promoter, prevents transcription • Allolactose made from lactose when it enters cell, lasts as long as lactose available – Inducer of lac operon by binding to lac repressor – Inducible operon because inducer increases expression a. Lactose absent from medium lac operon lacI Promoter Operator lacZ lacY lacA DNA Transcription blocked mRNA Lac repressor (active) RNA polymerase cannot bind to promoter When lactose is absent from the medium, the active Lac repressor binds to the operator of the lac operon, blocking transcription. Fig. 16-3a, p. 332 b. Lactose present in medium lac operon lacI Promoter Operator DNA mRNA Lac repressor (active) RNA polymerase binds and transcribes operon Binding site for inducer Allolactose (inducer) lacZ lacY lacA Transcription occurs Inactive repressor Translation Lactose metabolism enzymes mRNA When lactose is present in the medium, some of it is converted to the inducer allolactose. Allolactose binds to the Lac repressor, inactivating it so that it cannot bind to the operator. This allows RNA polymerase to bind to the promoter, and transcription of the lac operon occurs. Translation of the mRNA produces the three lactose metabolism enzymes. Fig. 16-3b, p. 332 Positive Regulation of lac Operon • lac operon operates when lactose but not glucose is present – Glucose more efficient energy source than lactose • Catabolite Activator Protein (CAP) is an activator that stimulates gene expression – CAP activated by cAMP – cAMP only abundant when glucose levels are low a. Lactose present; glucose low or absent CAP site Promoter Operator Transcription occurs lacZ lacI DNA cAMP CAP mRNA mRNA RNA polymerase Translation Active CAP Lac repressor (active) Allolactose (inducer) Lactose metabolism enzymes Inactive repressor When lactose is present and glucose is low or absent, cAMP levels are high. cAMP binds to CAP, activating it. Active CAP binds to the CAP site and recruits RNA polymerase to the promoter. Transcription then occurs. Fig. 16-5a, p. 334 a. Lactose present; glucose low or absent CAP site Promoter Operator Transcription occurs lacZ lacI DNA cAMP CAP mRNA mRNA RNA polymerase Active CAP Lac repressor (active) Allolactose (inducer) Translation Lactose metabolism enzymes Inactive repressor When lactose is present and glucose is low or absent, cAMP levels are high. cAMP binds to CAP, activating it. Active CAP binds to the CAP site and recruits RNA polymerase to the promoter. Transcription then occurs. Stepped Art Fig. 16-5a, p. 334 b. Lactose present; glucose present lacI CAP site Promoter Operator No transcription lacZ DNA RNA polymerase binding site mRNA Inactive CAP RNA polymerase cannot bind Lac repressor (active) Allolactose (inducer) Inactive repressor When lactose is present and glucose is present, cAMP levels are low. As a result, CAP is inactive and cannot bind to the CAP site. RNA polymerase then is unable to bind to the promoter, and no transcription occurs. Fig. 16-5b, p. 334 Summary of lac operon • Turn off unless Lactose is present (lac I protein active) • Turn on if Lactose is present (Lac I binding to allolactose; inactive) • Turn on if Lactose is present (CAMP binds to CAP to activate • Turn off again if Lactose AND Glucose are present (CAMP not available with glucose present; cannot activate CAP) Tryptophan metabolism • No Tryp available, the cell makes it ownthe operon is turned “on” • If tryp is available, the cell does not want the enzymes for synthesis to be made, so the cell turns it off • This is negative regulation – turning off rather than on a. Tryptophan absent from medium Regulatory RNA polymerase binds and transcribes operon gene trpR Promoter Operator trpE trp operon trpD trpC trpB trpA DNA mRNA Trp repressor (inactive) Transcription occurs mRNA Translation Tryptophan biosynthesis enzymes When tryptophan is absent from the medium, the Trp repressor is inactive in binding to the operator and transcription proceeds. Fig. 16-4a, p. 333 b. Tryptophan present in medium trp operon trpR Promoter Operator trpE trpD trpC trpB trpA DNA Transcription blocked mRNA Trp repressor (inactive) RNA polymerase cannot bind to promoter Tryptophanbinding site Tryptophan (corepressor) Trp repressor (active) When tryptophan is present in the medium, the amino acid binds to, and activates, the Trp repressor. The active repressor binds to the operator and blocks transcription. Fig. 16-4b, p. 333 Eukaryotic Transcription Regulation • In eukaryotes, regulation of gene expression occurs at several levels • Chromatin structure plays an important role in whether a gene is active or inactive • Regulation of transcription initiation involves a gene’s promoter and regulatory sites • Methylation of DNA can control gene transcription Regulation of Gene Expression in Eukaryotes • Gene expression in eukaryotes has more regulatory points – Chromatin has histones – Different types of cells – Nuclear envelope • Three main areas of eukaryotic regulation of gene expression – Transcriptional, posttrascriptional and posttranslational Chapter 16: Gene regulation • Diagram the lac operon transcription unit • Compare and contrast the operon model of tryptophan and lactose metabolism • Compare and contrast prokaryotic and eukaryotic gene regulation