Chapters 19-20 outline
Chapters 19-20 outline BIOSC 0160
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Chapter 19 Control of Gene Expression in Eukaryotes l Gene Regulation in Eukaryotes An Overview A Differential gene expression is responsible for creating different cell types arranging them into tissues and coordinating their activity to form the multicellular society we call an individual Eukaryotes can control gene expression at the levels of transcription translation and posttranslation 1 Three additional levels of control occur in eukaryotes a Chromatin remodeling The process by which the DNA in chromatin the complex of DNA and proteins mainly histones is unwound from its associated proteins to allow transcription or replication i It may involve chemical modi cation of histone proteins or reshaping of the chromatin by large multiprotein complexes in an ATPrequiring process RNA processing the steps required to produce a mature processed mRNA from a primary RNA transcript mRNA life span is regulated in eukaryotes ones that remain in the cell for a long time tend to be translated more than mRNAs that have a short life span ll Chromatin Remodeling A For a molecular signal to trigger the transcription of a particular gene the chromatin around the target gene must be remodeled 1 To t inside the nucleus the DNA must be packed tightly B so tightly that RNA polymerase can t access it What Is Chromatin s Basic Structure 1 2 Chromatin consists of DNA complexed with histones one of several positively charged basic proteins associated with DNA in the chromatin of eukaryotic cells and other proteins A nucleosome is a repeating unit of chromatin consisting of DNA wrapped twice around eight histone proteins a The intimate association between DNA and histones occurs in part because DNA is negatively charged and histones are positively charged i Histones are positively charged because they contain many lysines and arginines two positively charged amino acids H1 histones interact with one another and with histones in other nucleosomes to produce a tightly packed structure 3 Chromatin structure also has profound implications for the control of gene expression C Evidence that Chromatin Structure ls Altered in Active Genes 1 The close physical interaction between DNA and histones must be altered for RNA polymerase to make contact with DNA Chromatin must be decondensed to expose the promoter so RNA polymerase can bind to it DNA in Condensed Chromatin ls Protected from DNase a DNases are enzymes that cut DNA i Some cleave DNA at random locations and these cannot cut ef ciently if DNA is tightly wrapped with proteins ii It only works ef ciently if DNA is decondensed Histone Mutants a There are mutant yeast cells that don t produce the usual complement of histones In these mutants many genes that are normally never transcribed are instead always transcribed at high levels b In their normal default state eukaryotic genes are turned off D Howls Chromatin Altered 1 2 DNA Methylation a A group of enzymes known as DNA methyl transferases add methyl groups CH3 to cytosine residues in DNA i In mammals the sequence recognized by these enzymes is a C next to a G in one strand on the DNA b DNA methylation is important because methylated sequences are recognized by proteins that trigger chromatin condensation c Actively transcribed genes usually have low levels of methylation near their promoters and nontranscribed genes usually have high levels Histone Modi cation a A large set of enzymes adds a variety of chemical groups to speci c amino acids of histone proteins i These include phosphate groups methyl groups short polypeptide chains and acetyl groups ii Addition of these groups to histones promotes condensed or decondensed chromatin depending on the speci c set of modi cations b The histone code hypothesis postulates that particular combinations of histone modi cations set the state of chromatin condensation for a particular gene c Histone acetyltransferases HATS add acetyl groups to the positively charged lysine residues in histones Histone deacetylases HDACs remove them d Acetylation of histones usually results in decondensed chromatin a state associated with active transcription i When HATs add acetyl groups the acetyl groups neutralize the positive charge on lysine and loosens the close association of nucleosomes with the negatively charged DNA ii The addition of acetyl groups also creates a binding site for other proteins the help open the chromatin iii When HDACs remove acetyl groups it leads to condensed chromatin 3 ChromatinRemodeling Complexes a Chromatinremodeling complexes harness the energy in ATP to reshape chromatin by causing nucleosomes to slide along the DNA or knock the histones completely off to open up stretches of DNA E Chromatin Modi cations Can Be Inherited 1 Epigenetic inheritance is the patterns of inheritance that are due to something other than differences in DNA sequences environment Muscle cells are different from brain cells because they have inherited different patters of DNA methylation and histone modi cation during their development 3 Chromatin remodeling must occur before transcription Initiating Transcription Regulatory Sequences and Regulatory Proteins A The promoter is a site in DNA where RNA polymerase binds to initiate transcription 1 2 Promoters in eukaryotes are more complex such as the TATA box Once a promoter that contains a TATA box has been exposed by chromatin remodeling the rst step in initiating transcription is binding of the TATAbinding protein TBP B PromoterProximal Elements Are Regulatory Sequences Near Promoter 1 2 ln bacteria genes that need to be regulated together are often clustered into a single operon and transcribed into a single mRNA Eukaryotes regulated genes together by using an activator protein that exerts positive control over all of the genes a Coregulated genes are not clustered together but instead share a regulatory DNA sequence that binds the same regulatory protein 3 Regulatory sequences like these that are located close to the promoter and bind regulatory proteins are promoter proximal elements a Unlike the promoter promoterproximal elements have sequences that are unique to speci c sets of genes b They furnish a mechanism for eukaryotic cells to express certain genes but not others C Enhancers Are Regulatory Sequences Far From the Promoter 1 Regulatory sequences that are far from the promoter and activate transcription are enhancers 2 Enhancers occur in all eukaryotes and have several key characteristics a Can be more than 100000 bases away from promoter b Can be located in introns or in UTRs on either the 5 or 3 side of gene Many types exist Most genes have more than one Usually have binding sites for more than one protein Can work even if their 5 gt 3 orientation is ipped or they are moved to a new location in vicinity of gene 3 When regulatory proteins called transcriptional activators bind to enhancers transcription begins a Thus enhancers are an element in positive control 4 Silencers are DNA sequences that repressors bind to in order to shut down transcription a Thus silencers are an element in negative control D The Role of Transcription Factors in Differential Gene Expression 1 Enhancers and silencers are binding sites for activators and repressors a These proteins are regulatory transcription factors b Different types of cells express different genes because they have different transcription factors E How Do Transcription Factors Recognize Speci c DNA Sequences 1 Differences in composition and shape can be recognized by transcription factors 2 Just a base pairs come together by complementary interactions so can proteins and speci c DNA sequences F A Model For Transcription Initiation 1 Basal transcription factors are proteins that interact with the promoter and are not restricted to particular genes or cell types rhme a These proteins are necessary for transcription to occur but they do not regulate b The TATAbinding protein is a BTF 2 A large complex of proteins called Mediator acts as a bridge between regulatory transcription factors basal transcription factors and RNA polymerase II 3 How transcription is initiated in eukaryotes a Transcriptional activators bind to DNA and recruit chromatinremodeling complexes and histone acetyltransferases b The chromatinremodeling complexes and HATs open a swatch of chromatin that includes the promoter promoterproximal elements and enhancers c Other transcriptional activators bind to the newly exposed enhancers and promoterproximal elements basal transcription factors bind to the promoter and recruit RNA polymerase II d Mediator connects the transcriptional activators and basal transcription factors that are bound to DNA This step is made possible through DNA looping 4 Activators work not only to stimulate transcription but also to bring chromatinremodeling proteins to the right place at the right time IV PostTranscriptionalControl A Once a gene is transcribed a series of events has to occur before a nal product appears These include 1 Splicing mRNAs in various ways 2 Modifying the life span of mRNAs 3 Altering the rate at which translation is initiated 4 Activating or inactivating proteins after translation has occured B Alternative Splicing of mRNAs 1 During splicing gene expression is regulated when selected exons are removed from the primary transcript along with the introns a As a result the same RNA transcript can yield more than one kind of mature mRNA consisting of different combinations of exons b This is referred to as alternative splicing 2 Proteins that bind to RNAs in the nucleus and interact with spliceosomes to in uence which sequences are used for splicing control alternative splicing a 90 of genes undergo alternative splicing C mRNA Stability and RNA lnterface 1 Once splicing is complete and process mRNAs are exported to the cytoplasm new regulatory mechanisms come into play 2 RNA interface occurs when a tiny singlestranded RNA held by a protein complex binds to a complementary sequence in an mRNA a This unleashes either the destruction of the mRNA or a block to the mRNA s translation 3 Sequence of events of RNA interface a Begins when RNA polymerase transcribes genes that code for RNAs that double back on themselves to form a hairpin i Hairpin formation occurs because pairs of bases within the RNA transcript are complementary Some of the RNA is trimmed by enzymes in the nucleus then the doublestranded hairpin that remains is exported to the cytoplasm In the cytoplasm another enzyme cuts out the hairpin loop to form doublestranded RNA molecules that are only about 22 nucleotides long One of the strands from this short RNA is taken up by a group of proteins called the RNAinduced silencing complex or RISC i The RNA strand held by the RISC is a microRNA miRNA Once it is part of a RISC the miRNA binds to its complementary sequences in a target mRNA If the match is perfect an enzyme in the RISC destroys the mRNA by cutting it in two If the match isn t perfect the mRNA is not destroyed but its translation is inhibited 4 miRNAs are critical for normal development and mutations in miRNA genes are associated with many diseases D Howls Translation Controlled 1 Cells may slow or stop translation in response to a sudden increase in temperature or infection by a virus a The slowdown occurs because regulatory proteins that are activated by the temperature spike or viral invasion add a phosphate group to a protein that is part of the ribosome i The phosphorylation causes a shape change which slows or prevents translation E PostTranslational Control 1 Posttranslation regulation allows cells to respond rapidly to new conditions 2 Instead of waiting for transcription RNA processing and translation to occur the cell can keep an existing but inactive protein waiting in the wings and then quickly activate it in response to altered conditions Speed is gained at the expense of efficiency Mechanisms Protein folding Addition of carbohydrate groups Cleaving off certain amino acids Phosphorylation Targeted destruction of proteins i Enzymes mark these proteins by adding many copies of Ubiquitin ii The proteasome recognizes these proteins and cuts them into short segments DP00quot V How Does Gene Expression Compare in Bacteria and Eukaryotes A Five fundamental differences 1 5 DNA packaging The default state of transcription in bacteria is quotonquot Because of histones in eukaryotes there is a mechanism of negative control that doesn t exist in bacteria Complexity of transcription The sheer number of eukaryotic proteins involved in regulating transcription is more than in bacteria Coordinated transcription Eukaryotes do not have regulons physically scattered genes are expressed together when the same regulatory transcription factors trigger the transcription of genes with the same DNA regulatory sequences Greater reliance on posttranscriptional control Alternative splicing allows eukaryotes to regulate the production of many proteins from each gene Alternative splicing microRNAs and regulation of mRNA stability are seldom seen in bacteria Vl Linking Cancer with Defects in Gene Regulation A Each type of cancer is caused by a different set of mutations that lead to cancer when they affect one of two classes of genes 1 2 Those that stop or slow the cell cycle Those that trigger cell growth and division by initiating speci c phases in the cell cycle B The Genetic Basis of Uncontrolled Cell Growth 1 Proteins that stop or slow the cell cycle when conditions are unfavorable for cell division are tumor suppressors a Genes that code for these proteins are tumor suppressor genes b If the function of this gene is lost because of mutation then a key brake on the cell cycle is eliminated 2 Genes that stimulate cell division are protooncogenes a In normal cells the proteins produced from these genes are active only when conditions are appropriate for growth b In cancer cells defects in the regulation of proto oncogenes can cause these genes to stimulate growth at all times 3 A mutation can convert the protooncogene to an oncogene an allele that promotes cancer development C The p53 Tumor Suppressor 1 The p53 gene codes for a regulatory transcription factor that serves as a master brake on the cell cycle a It is most often defective in cancer cells b P53 is activated by DNA damage 2 Activated p53 binds to the enhancers of genes that arrest the cell cycle repair DNA damage and trigger apoptosis 3 In mutant cells that lack a form of p53 that can bind to enhancers DNA damage cannot arrest the cell cycle the cell cannot kill itself and damaged DNA is replicated Chapter 20 Analyzing and Engineering Genes HGH Basic Recombinant DNA Technologies A The pituitary gland produces several important biomolecules including HCH a protein that stimulates growth which is coded for by the gene GHJ 1 Some forms of inherited dwar sm are caused by a defect in the GHJ gene B Why Did Early Efforts To Treat the Disease Fail 1 To treat the disease early physicians injected naturally produced growth hormone to those affected a This only worked if the protein came from humans b The only source of HGH was pituitary glands from human cadavers i Contaminated with a prion protein from cadavers banned C Steps in Engineering a Safe Supply of HGH 1 Researchers planned to insert fully functional copies of human GHl into E Coiwhich they hoped would then produce huge quantities of the recombinant progeny 2 Using Reverse Transcriptase to Produce cDNAs a b Reverse transcriptase catalyzes the synthesis of DNA from an RNA template DNA produced from RNA is called complementary DNA or cDNA i Although reverse transcriptase initially produces a sscDNA it is also capable of synthesizing the complementary strand to yield dsDNA Knowing GHJ is actively transcribed in the pituitary gland researchers isolated mRNAs from pituitary gand cells and used the enzyme to reverse transcribe those mRNAs to cDNAs i The products contained dscDNAs corresponding to each gene that is actively expressed in pituitary cells 3 Using Plasmids in Cloning a b Producing many copies of a gene is DNA cloning In many cases researchers can clone a gene by inserting into a plasmid C Researchers realized that if they could splice a loose piece of DNA into a plasmid and then insert the modi ed plasmid into a bacterial cell the engineered plasmid would be replicated and passed on to daughter cells i When a plasmid is used this way its called a cloning vector d The recombinant genes are harvested by breaking the bacteria open isolating the DNA and then separating the plasmids from the main chromosomes Using Restriction Endonucleases and DNA Ligase to Cut and Paste a To cut a gene out for later insertion into a cloning vector restriction endonucleases bacterial enzymes that cut DNA molecules at speci c base sequences are used To insert the pituitarygland cDNAs into plasmids I Identify a palindromic recognition site ii Add restriction endonuclease to cut recognition sites in each plasmid and at the ends of each cDNA iii Sticky ends result and are capable of hydrogen bonding with a complementary sequence iv lnsert gene into plasmid using DNA ligase Transformation Introducing Plasmids into Bacterial Cells a If a recombinant plasmid can be inserted into a bacterial or yeast cell the foreign DNA will be copied and transmitted to new cells as the host cell grows and divides cloning Cells that take up DNA from the environment and incorporate it into their genomes are said to undergo transformation Producing a cDNA Library a The result is a collection of transformed bacterial cells that each contain a plasmid with one cDNA from a pituitary gland mRNA b A collection of DNA sequences each of which is inserted into a vector is called a DNA library If the sequences are cDNAs the library is a cDNA library If the sequences are fragments of DNA from the genome of an individual it is a genomic library DNA libraries are important because they provide researchers a way to store DNA fragments from a particular cell type of genome in a form that is accessible for gene cloning 7 Screening a DNA Library a To nd one speci c gene in a large collection of DNA fragments a probe a marked molecule that binds to the molecule the biologist is looking for is required b A DNA probe is a singlestranded fragment that will bind to a particular singlestranded complementary sequence in a mixture of DNAs By binding to the target sequence the probe marks the fragment contain that sequence distinguishing it from other DNA fragments in the sample c Steps i Grow transformed cells containing plasmids on many plates Each colony contains a different cDNA ii Lay a lter on each plate then remove iii Treat bacteria with chemicals to break open cells and make DNAs single stranded iv Probe lters with labeled DNA v Find probe vi Identify colony 8 MassProducing Growth Hormone a Researchers used recombinant DNA techniques to transfer the growth hormone cDNA to new plasmid that contained a promoter sequence that is recognized by E coli s RNA polymerase holoenzyme b The resulting transformed E coli cells contained a gene for HGH attached to a promoter i They began to transcribe and translate the HGH gene ii HGH accumulated in the cells and was isolated D Ethical Concerns Over Recombinant Growth Hormone 1 Used as a performanceenhancing drug
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