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 right- handed 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

Fig. 14-4, p ' end 3' end Deoxyribose (a 5-carbon sugar) Phosphate Adenine (A) Guanine (G) Thymine (T) Cytosine (C) Purines (double-ring structures) Pyrimidines (single-ring structures) Hydroxyl group

Fig. 14-6, p nm 5' end Distance between each pair of bases = 0.34 nm Each full twist of the DNA double helix = 3.4 nm Phosphate group 5-carbon sugar deoxyribose) Nitrogenous base (guanine) Hydrogen bond 5' end 3' end

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

Fig. 14-8a, p. 285 Parental DNA Replicated DNA KEY a. Semiconservative replication 2nd replication 1st 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.

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)

Fig , p. 291 Chromosome strand shortened 3' end of template strand PrimerNew DNA Gap left by primer removal Primer removed. 3' end of DNA template unwound and ready for replication. 1 Primer added and new DNA assembled from end of primer. 2 3

Fig , p Original end of chromosome Added telomere repeats Primer added to chromosome end Primer removed Gap filled in Chromosome strand not shortened Extra telomere repeats added by telomerase at 3’ end of template strand Primer added and gap filled in Primer removed; original length is restored 1 2

DNA Synthesis Begins at sites that act as replication origins Proceeds from the origins as two replication forks moving in opposite directions

Fig , p. 292 Replication forks OriginDNA double helix Replication direction

Fig , p. 297 DNA double helix Origin Replication forks

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

Fig , p. 293 New strand Template strandDNA polymerase Enzyme continues activity in the forward direction as DNA 3’ polymerase as long as the most recently added nucleotide is correctly paired. 1 4 Enzyme adds a mispaired nucleotide. Enzyme reverses, acting as a deoxyribonuclease to remove the mispaired nucleotide. Enzyme resumes forward activity as a DNA polymerase. 2 3 New strand

Fig , p. 293 Base-pair mismatch Nick left after gap filled in Template strand New strand 4 Repair enzymes recognize a mispaired base and break one chain of the DNA at the arrows. The enzymes remove several to many bases, including the mismatched base, leaving a gap in the DNA. The gap is filled in by a DNA polymerase using the intact template strand as a guide. The nick left after gap filling is sealed by DNA ligase to complete the repair

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

Fig. 16-2, p. 331 lacY DNA Lac repressor Regulatory gene lacI Binds Lac repressor Binds RNA polymerase Transcription initiation site Promoter Operator Sequences that control the expression of the operon β-Galactosidase lacZ lac operon PermeaseTransacetylase Transcription termination site lacA

lac Operon for Lactose Metabolism lac repressor stops lac operon expression –Encoded by lac I, 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

Fig. 16-3a, p. 332 lacA DNA mRNA Lac repressor (active) lacI a. Lactose absent from medium RNA polymerase cannot bind to promoter Promoter Operator Transcription blocked When lactose is absent from the medium, the active Lac repressor binds to the operator of the lac operon, blocking transcription. lac operon lacZ lacY

Fig. 16-3b, p. 332 Transcription occurs b. Lactose present in medium DNA Lac repressor (active) mRNA Binding site for inducer Allolactose (inducer) RNA polymerase binds and transcribes operon lacI PromoterOperator 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. Lactose metabolism enzymes lacA mRNA Translation lacY lac operon lacZ Inactive repressor

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

Fig. 16-5a, p. 334 RNA polymerase DNA a. Lactose present; glucose low or absent mRNA Lac repressor (active) Allolactose (inducer) Inactive repressor lacI cAMP CAP site CAP Active CAP Promoter 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. Operator Transcription occurs lacZ Translation Lactose metabolism enzymes mRNA

DNA lacI CAP sitePromoterOperator lacZ Fig. 16-5a, p. 334 a. Lactose present; glucose low or absent mRNA Lac repressor (active) Allolactose (inducer) Inactive repressor cAMP CAP Active CAP Transcription occurs Translation Lactose metabolism enzymes mRNA RNA polymerase Stepped Art 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. 16-5b, p. 334 RNA polymerase binding site b. Lactose present; glucose present DNA mRNA Lac repressor (active) Allolactose (inducer) Inactive repressor Inactive CAP CAP sitePromoter Operator No transcription RNA polymerase cannot bind lacIlacZ 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.

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 own- the 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

Fig. 16-4a, p. 333 mRNA DNA a. Tryptophan absent from medium Trp repressor (inactive) mRNA Regulatory gene trpR RNA polymerase binds and transcribes operon Promoter Operator trpE Transcription occurs When tryptophan is absent from the medium, the Trp repressor is inactive in binding to the operator and transcription proceeds. trp operon trpD trpCtrpBtrpA Tryptophan biosynthesis enzymes Translation

Fig. 16-4b, p. 333 trpE trpD trpC trpBtrpA trpR Promoter Operator trp operon b. Tryptophan present in medium Transcription blocked DNA mRNA Trp repressor (inactive) Tryptophan (corepressor) RNA polymerase cannot bind to promoter Trp repressor (active) Tryptophan- binding site 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.

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