Eukaryotic Genome Organization & Regulation PDF

Concept 1: Chromatin structure is based on successive levels of DNA packing Prokaryotic and eukaryotic cells both contain double-stranded DNA, but their genomes...

Concept 1: Chromatin structure is based on successive levels of DNA packing Prokaryotic and eukaryotic cells both contain double-stranded DNA, but their genomes are organized differently: Eukaryotic Genomes: Prokaryotic DNA is: Organization & Regulation Usually circular Much smaller than eukaryotic DNA; it makes up a small nucleoid region only visible with an electron microscope Associated with only a few protein molecules Presented by Dr Ghada Khawaja Less elaborately structured and folded than eukaryotic DNA Eukaryotic DNA is: Complexed with a large amount of protein to form chromatin Highly extended and tangled during interphase Condensed into short, thick, discrete chromosomes during mitosis; when stained, chromosomes are clearly visible with a light microscope Eukaryotic chromosomes contain an enormous amount of DNA, which requires an elaborate system of DNA packing to fit all of the cell's DNA into the nucleus Overview: How Eukaryotic Genomes Work and Evolve Nucleosomes, or “Beads on a String” In eukaryotes, the DNA-protein complex, called Proteins called histones chromatin – Are responsible for the first level of DNA packing in chromatin – Is ordered into higher structural levels than the DNA- – Bind tightly to DNA protein complex in prokaryotes The association of DNA and histones – Seems to remain intact throughout the cell cycle Histones = Small proteins that are rich in basic amino acids and that bind to DNA, forming chromatin. Contain a high proportion of positively charged amino acids (arginine and lysine), which bind tightly to the negatively charged DNA Are present in approximately equal amounts to DNA in eukaryotic cells Are similar from one eukaryote to another, suggesting that histone genes have been highly conserved during evolution. There are five types of histones in eukaryotes.  In electron micrographs – Unfolded chromatin (DNA and its associated proteins) has the appearance of beads on a DNA string Nucleosomes may control gene expression by Each “bead” is a nucleosome: controlling access of transcription proteins to DNA – The basic unit of DNA packing; it is formed from DNA wound around a protein core that consists of two copies each of four types of histone (H2A, H2B, H3, H4). – A fifth histone (H1) attaches near the bead when the chromatin undergoes the Nucleosome heterogeneity may also help control next level of packing gene expression; nucleosomes may differ in the extent of amino acid modification and in the type of 2 nm nonhistone proteins present. DNA double helix His- Histone tones tails Histone H1 10 nm Linker DNA Nucleosome (“string”) (“bad”) (a) Nucleosomes (10-nm fiber) Higher Levels of DNA Packing The 30-nm fiber, in turn The next level of packing – Forms looped domains which: are attached to a nonhistone protein scaffold – Forms the 30-nm chromatin fiber making up a 300-nm fiber Protein scaffold Loops 30 nm 300 nm Scaffold Nucleosome (c) Looped domains (300-nm fiber) (b) 30-nm fiber This structure consists of a tightly wound coil with six nucleosomes per turn. Molecules of histone H1 pull the nucleosomes into a cylinder 30nm in diameter  In a mitotic chromosome Concept 2: Gene expression can be regulated at – The looped domains themselves coil and fold any stage, but the key step is transcription forming the characteristic metaphase chromosome All organisms – Must regulate which genes are expressed at any given time During development of a multicellular organism 700 nm – Its cells undergo a process of specialization in form and function called cell differentiation 1,400 nm Cell differentiation requires that gene expression must be regulated (d) Metaphase chromosome Highly specialized cells, such as muscle or nerve, express only a small percentage of their genes, so transcription enzymes must locate the right genes at the right In interphase cells time. Uncontrolled or incorrect gene action can cause serious imbalances and disease, including cancer. Thus, eukaryotic gene regulation is of interest in medical as well as basic research. – Most chromatin is in the highly extended form called euchromatin (much less condensed than mitotic chromatin) Differential Gene Expression Portions of some chromosomes remain highly condensed throughout the cell cycle, even during interphase. Such heterochromatin is not transcribed. Each cell of a multicellular eukaryote Heterochromatin = Chromatin that remains highly condensed during interphase – Expresses only a fraction of its genes and that is not actively transcribed In each type of differentiated cell Euchromatin = Chromatin that is less condensed during interphase and is actively transcribed; euchromatin becomes highly condensed during mitosis – A unique subset of genes is expressed What is the function of heterochromatin in interphase cells? Since most heterochromatin is not transcribed, it may be a coarse control of gene expression.  Histone Modification Many key stages of gene expression Chemical modification of histone tails – Can be regulated in eukaryotic cells Signal – Can affect the configuration of chromatin and thus NUCLEUS Chromatin gene expression Chromatin modification: DNA unpacking involving histone acetylation and Chromatin changes DNA demethlation DNA Gene available for transcription Transcription Gene Transcription RNA processing RNA Exon Primary transcript Intron RNA processing mRNA Translation Tail degradation Cap mRNA in nucleus Protein processing Transport to cytoplasm and degradation CYTOPLASM Histone mRNA in cytoplasm tails Degradation of mRNA Translation Polypetide Cleavage Chemical modification Transport to cellular DNA destination double helix Amino acids Active protein available Degradation of protein for chemical modification Degraded protein (a) Histone tails protrude outward from a nucleosome Histone acetylation – Seems to loosen chromatin structure and thereby 1/ Regulation of Chromatin enhance transcription (enhance the access of Structure transcription proteins to DNA) Chromatin modifications affect the availability of genes for transcription Unacetylated histones Acetylated histones Genes within highly packed heterochromatin (b) Acetylation of histone tails promotes loose chromatin structure that permits transcription – Are usually not expressed  Organization of a Typical Eukaryotic Gene DNA Methylation Associated with most eukaryotic genes are multiple control elements: Addition of methyl groups to certain bases – Segments of noncoding DNA that help regulate in DNA transcription by binding certain proteins (transcription – Is associated with reduced transcription in some factors) Enhancer Proximal Poly-A signal Termination (distal control elements) control elements sequence region species Exon Intron Exon Intron Exon DNA Upstream Downstream Promoter Transcription Poly-A signal Primary RNA Exon Intron Exon Intron Exon Cleared 3 end transcript 5 of primary Chromatin changes (pre-mRNA) transport RNA processing: Transcription Cap and tail added; introns excised and Intron RNA exons spliced together RNA processing mRNA Translation Coding segment degradation Protein processing and degradation mRNA G P P P 5 Cap 5 UTR Start Stop 3 UTR Poly-A codon codon (untranslated tail (untranslated region) region) 2/ Regulation of Transcription The Roles of Transcription Factors Initiation To initiate transcription – Eukaryotic RNA polymerase requires the assistance Transcription initiation is controlled of proteins called transcription factors by proteins that interact with DNA and with each other Chromatin-modifying enzymes provide initial control of gene expression – By making a region of DNA either more or less able to bind the transcription machinery  An activator – Is a protein that binds to an enhancer and stimulates transcription of a gene Distal control element Promoter Activators Gene Enhancer TATA box General 1 Activator proteins bind to distal control elements transcription grouped as an enhancer in factors the DNA. This enhancer has DNA-bending three binding sites. protein 2 A DNA-bending protein Group of brings the bound activators Mediator proteins closer to the promoter. Other transcription factors, RNA mediator proteins, and RNA Polymerase II polymerase are nearby. Chromatin changes 3 The activators bind to Transcription certain general transcription RNA processing factors and mediator RNA proteins, helping them form mRNA Translation Polymerase II degradation an active transcription Protein processing initiation complex on the promoter. and degradation Transcription RNA synthesis Initiation complex Enhancers and Specific Transcription Some specific transcription factors function as repressors Factors – To inhibit expression of a particular gene Proximal control elements Some activators and repressors – Are located close to the promoter – Act indirectly by influencing chromatin structure Distal control elements, groups of which are called enhancers – May be far away from a gene or even in an intron  Coordinately Controlled Genes RNA Processing In alternative RNA splicing Coordinately controlled genes are arranged differently in a eukaryotic than in prokaryotic genomes. Prokaryotic genes that are turned on and off together are often clustered into operons; these – Different mRNA molecules are produced from the same adjacent genes share regulatory sites located at one end of the cluster. All genes of the primary transcript, depending on which RNA segments operon are transcribed into one mRNA molecule and are translated together. are treated as exons and which as introns Chromatin changes Eukaryotic genes coding for enzymes of a metabolic pathway are often scattered over Transcription different chromosomes. Even functionally related genes on the same chromosome have their RNA processing mRNA Translation own promoters and are individually transcribed. degradation Protein processing and degradation Eukaryotic genes can be coordinately expressed, even though they may be scattered Exons throughout the genome. DNA Coordinately controlled genes are each associated with specific regulatory DNA Primary RNA transcript sequences or enhancers. These sequences are recognized by a single type of RNA splicing or transcription factor that activates or represses a group of genes in synchrony. mRNA 3/ Mechanisms of Post- mRNA Degradation Transcriptional Regulation The life span of mRNA molecules in the cytoplasm – Is an important factor in determining the protein Post-Transcriptional mechanisms play supporting roles in the control of gene expression synthesis in a cell – Is determined in part by sequences in the leader and trailer regions An increasing number of examples – Are being found of regulatory mechanisms that operate at various stages after transcription  RNA interference by single-stranded microRNAs (miRNAs) Protein Processing and Degradation – Can lead to degradation of an mRNA or block its translation After translation 1 The micro- 2 An enzyme 2 3 One strand of 4 The bound 5 The miRNA-protein 5 – Various types of protein processing, including RNA (miRNA) called Dicer moves each short double- miRNA can base-pair complex prevents gene precursor folds back on itself, along the double- stranded RNA, stranded RNA is degraded; the other with any target mRNA that contains expression either by degrading the target cleavage and the addition of chemical groups, are held together cutting it into strand (miRNA) then the complementary mRNA or by blocking by hydrogen bonds. shorter segments. associates with a complex of proteins. sequence. its translation. subject to control Chromatin changes Transcription RNA processing mRNA Translation degradation Protein processing Protein and degradation complex Dicer Degradation of mRNA OR miRNA Target mRNA Hydrogen Blockage of translation bond Initiation of Translation Proteasomes – Are giant protein complexes that bind protein The initiation of translation of selected molecules and degrade them mRNAs 3 Enzymatic components of the 2 The ubiquitin-tagged protein proteasome cut the protein into – Can be blocked by regulatory proteins that bind to 1 Multiple ubiquitin mol- ecules are attached to a protein is recognized by a proteasome, which unfolds the protein and small peptides, which can be further degraded by other by enzymes in the cytosol. sequesters it within a central cavity. enzymes in the cytosol. specific sequences or structures of the mRNA Chromatin changes Transcription Alternatively, translation of all the mRNAs RNA processing Proteasome Ubiquitin in a cell mRNA Translation degradation and ubiquitin to be recycled Protein processing Proteasome and degradation – May be regulated simultaneously Protein to Ubiquinated Protein be degraded protein fragments (peptides) Protein entering a proteasome  Concept 3: Cancer results from genetic changes Oncogenes and Proto-Oncogenes that affect cell cycle control The gene regulation systems that go wrong Oncogenes during cancer – Are cancer-causing genes – Turn out to be the very same systems that play Proto-oncogenes important roles in embryonic development – Are normal cellular genes that code for proteins that stimulate normal cell growth and division Types of Genes Associated with A DNA change that makes a proto-oncogene Cancer excessively active – Converts it to an oncogene, which may promote The genes that normally regulate cell growth excessive cell division and cancer and division during the cell cycle – Include genes for growth factors, their receptors, Proto-oncogene DNA and the intracellular molecules of signaling pathways Translocation or transposition: gene moved to new locus, Gene amplification: Point mutation within a control Point mutation within the gene under new controls multiple copies of the gene element New Oncogene Oncogene promoter Normal growth-stimulating Normal growth-stimulating Normal growth-stimulating Hyperactive or protein in excess protein in excess protein in excess degradation- resistant protein  Tumor-Suppressor Genes The Ras protein, encoded by the ras gene – Is a G protein that relays a signal from a growth Tumor-suppressor genes factor receptor on the plasma membrane to a – Encode proteins that inhibit abnormal cell division cascade of protein kinases 1 Growth MUTATION factor Hyperactive Ras Ras protein GTP (product of 3 G protein oncogene) (a) Cell cycle–stimulating pathway. issues signals This pathway is triggered by 1 a growth Ras on its own P factor that binds to 2 its receptor in the P P P GTP plasma membrane. The signal is relayed to 3 P P a G protein called Ras. Like all G proteins, Ras is active when GTP is bound to it. Ras passes the signal to 4 a series of protein kinases. 4 Protein kinases The last kinase activates 5 a transcription 2 Receptor (phosphorylation activator that turns on one or more genes cascade) for proteins that stimulate the cell cycle. If a NUCLEUS mutation makes Ras or any other pathway component abnormally active, excessive cell 5 Transcription division and cancer may result. factor (activator) DNA Gene expression Protein that stimulates the cell cycle Interference with Normal Cell- The p53 gene encodes a tumor-suppressor protein Signaling Pathways – That is a specific transcription factor that promotes the synthesis of cell cycle–inhibiting proteins Many proto-oncogenes and tumor suppressor genes – Encode components of growth-stimulating and (b) Cell cycle–inhibiting pathway. In this pathway, 1 DNA damage is an intracellular signal that is passed via 2 protein kinases and leads to activation of 3 p53. Activated 2 Protein kinases MUTATION growth-inhibiting pathways, respectively p53 promotes transcription of the gene for a protein that inhibits the cell cycle. The resulting suppression of cell division ensures Defective or missing transcription that the damaged DNA is not replicated. factor, such as Mutations causing deficiencies in any UV p53, cannot pathway component can contribute to the 3 Active light activate development of cancer. form transcription of p53 1 DNA damage DNA in genome Protein that inhibits the cell cycle  Mutations that knock out the p53 gene A multistep model for the development of – Can lead to excessive cell growth and cancer colorectal cancer Colon (c) Effects of mutations. Increased cell division, possibly leading to cancer, EFFECTS OF MUTATIONS can result if the cell cycle is 1 Loss of tumor- overstimulated, as in (a), or not Protein suppressor 4 Loss of Protein absent inhibited when it normally would be, overexpressed Colon wall gene APC (or 2 Activation of tumor-suppressor as in (b). ras oncogene other) gene p53 Cell cycle Increased cell Cell cycle not 3 Loss of 5 Additional overstimulated division inhibited tumor- mutations Normal colon Small benign suppressor Larger benign Malignant tumor epithelial cells growth (polyp) gene DCC growth (adenoma) (carcinoma) The Multistep Model of Cancer Certain viruses Development – Promote cancer by integration of viral DNA into a cell’s genome Normal cells are converted to cancer cells – By the accumulation of multiple mutations affecting proto-oncogenes and tumor-suppressor genes  Inherited Predisposition to Cancer Individuals who inherit a mutant oncogene or tumor-suppressor allele – Have an increased risk of developing certain types of cancer