Eukaryotic Genomes

Eukaryotic Genomes In eukaryotes, the DNA-protein complex, called chromatin is ordered into higher structural levels than the DNA-protein complex in prokaryotes Chromatin structure is based on successive levels of DNA packing Eukaryotic DNA is precisely combined with a large amount of protein Eukaryotic chromosomes contain an enormous amount of DNA relative to their condensed length

Nucleosomes: “Beads on a String” Proteins called histones are responsible for the first level of DNA packing in chromatin Histones bind tightly to DNA and their association seems to remain intact throughout the cell cycle In electron micrographs unfolded chromatin has the appearance of beads on a string Each “bead” is a nucleosome - the basic unit of DNA packing 2 nm 10 nm DNA double helix Histone tails His- tones Linker DNA (“string”) Nucleosome (“bead”) Histone H1 (a) Nucleosomes (10-nm fiber)

Higher Levels of DNA Packing The next level of packing forms the 30-nm chromatin fiber The 30-nm fiber, in turn forms looped domains, making up a 300-nm fiber Nucleosome 30 nm (b) 30-nm fiber Protein scaffold 300 nm (c) Looped domains (300-nm fiber) Loops Scaffold

Chromatin Condensation In interphase cells most chromatin is in the highly extended form called euchromatin In a mitotic chromosome the looped domains themselves coil and fold forming the characteristic metaphase chromosome 700 nm 1,400 nm (d) Metaphase chromosome

Eukaryotic Gene Regulation Signal NUCLEUS Chromatin Chromatin modification: DNA unpacking involving histone acetylation and DNA demethlation Gene DNA Gene available for transcription RNA Exon Transcription Primary transcript RNA processing Transport to cytoplasm Intron Cap mRNA in nucleus Tail CYTOPLASM mRNA in cytoplasm Degradation of mRNA Translation Polypetide Cleavage Chemical modification Transport to cellular destination Active protein Degradation of protein Degraded protein In eukaryotes, gene regulation is more complex Many key stages of gene expression can be regulated in eukaryotic cells However, transcriptional controls are still the primary method of gene regulation But there are also posttranscriptional controls.

Eukaryotic Gene Regulation - Transcription Chromatin modification Histone modification DNA methylation Transcription factors and control Elements Activators Repressors

Eukaryotic Gene Regulation - Transcripton Coiling of DNA within the nucleus help regulate gene transcription in eukaryotes. Studies have shown that transcription factors are unable to bind to promoters located in regions of DNA that are coiled around the histones of a nucleosome: 2 nm 10 nm DNA double helix Histone tails His- tones Linker DNA (“string”) Nucleosome (“bead”) Histone H1 (a) Nucleosomes (10-nm fiber)

Chromatin Modification 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 Chemical modification of histone tails can affect the configuration of chromatin and thus gene expression (a) Histone tails protrude outward from a nucleosome Chromatin changes Transcription RNA processing mRNA degradation Translation Protein processing and degradation DNA double helix Amino acids available for chemical modification Histone tails

Histone Modification Histone acetylation (COCH3)seems to loosen chromatin structure and thereby enhance transcription Figure 19.4 b (b) Acetylation of histone tails promotes loose chromatin structure that permits transcription Unacetylated histones Acetylated histones

DNA Methylation Addition of methyl groups to certain bases in DNA is associated with reduced transcription

Eukaryotic Gene Regulation – Control Elements Associated with most eukaryotic genes are multiple control elements - segments of noncoding DNA that help regulate transcription by binding certain proteins Distal control elements, groups of which are called enhancers may be far from a gene Regulatory molecules called activators can bind to regulatory enhancers to facilitate transcription factors Enhancer (distal control elements) Proximal control elements DNA Upstream Promoter Exon Intron Poly-A signal sequence Termination region Transcription Downstream Poly-A signal Primary RNA transcript (pre-mRNA) 5 Intron RNA RNA processing: Cap and tail added; introns excised and exons spliced together Coding segment P G mRNA 5 Cap 5 UTR (untranslated region) Start codon Stop 3 UTR tail Chromatin changes RNA processing degradation Translation Protein processing and degradation Cleared 3 end of primary transport

Transcription Factors RNA PROCESSING TRANSLATION DNA Pre-mRNA mRNA Ribosome Polypeptide T A TATA box Start point Template DNA strand 5 3 Transcription factors Promoter RNA polymerase II Transcription factors RNA transcript Transcription initiation complex Eukaryotic promoters 1 Several transcription 2 Additional transcription 3 To initiate transcription, eukaryotic RNA polymerase requires the assistance of proteins called transcription factors General transcription factors are essential for the transcription of all protein-coding genes. Only a few general transcription factors independently bind to a DNA sequence such as the TATA box within the promoter. Others in the initiation complex are involved in protein-protein interactions, binding each other and RNA polymerase II.

Regulation of Transcription Initiation In eukaryotes, regulatory molecules called activators can bind to regulatory enhancers Distal control element Activators Enhancer Promoter Gene TATA box General transcription factors DNA-bending protein Group of Mediator proteins RNA Polymerase II RNA synthesis Transcription Initiation complex Chromatin changes RNA processing degradation mRNA Translation Protein processing and degradation A DNA-bending protein brings the bound activators closer to the promoter. Other transcription factors, mediator proteins, and RNA polymerase are nearby. 2 Activator proteins bind to distal control elements grouped as an enhancer in the DNA. This enhancer has three binding sites. 1 The activators bind to certain general transcription factors and mediator proteins, helping them form an active transcription initiation complex on the promoter. 3

Combinatorial Control of Gene Activation A particular combination of control elements will be able to activate transcription only when the appropriate activator proteins are present Enhancer Promoter Control elements Albumin gene Crystallin Liver cell nucleus Lens cell Available activators expressed gene not Crystallin gene not expressed (a) (b)

Repressors Some specific transcription factors function as repressors proteins inhibit expression of a particular gene Eukaryotic repressors can cause inhibition of gene expression by blocking the binding of activators to their control elements or to components of the transcription machinery or by turning off transcription even in the presence of activators.

Post-transcriptional control Although less common than transcriptional control of gene expression, various types of posttranscriptional control may also occur in eukaryotes An increasing number of examples are being found of regulatory mechanisms that operate at various stages after transcription mRNA splicing mRNA degradation – miRNA Protein degradation

RNA Processing In alternative RNA splicing different mRNA molecules are produced from the same primary transcript, depending on which RNA segments are treated as exons and which as introns Chromatin changes Transcription RNA processing mRNA degradation Translation Protein processing and degradation Exons DNA Primary RNA transcript RNA splicing or

Blockage of translation mRNA Degradation RNA interference by single-stranded microRNAs (miRNAs) can lead to degradation of an mRNA or block its translation 5 Chromatin changes Transcription RNA processing mRNA degradation Translation Protein processing and degradation Degradation of mRNA OR Blockage of translation Target mRNA miRNA Protein complex Dicer Hydrogen bond The micro- RNA (miRNA) precursor folds back on itself, held together by hydrogen bonds. 1 2 An enzyme called Dicer moves along the double- stranded RNA, cutting it into shorter segments. One strand of each short double- stranded RNA is degraded; the other strand (miRNA) then associates with a complex of proteins. 3 The bound miRNA can base-pair with any target mRNA that contains the complementary sequence. 4 The miRNA-protein complex prevents gene expression either by degrading the target mRNA or by blocking its translation.

Post translation After translation various types of protein processing, including cleavage and the addition of chemical groups, are subject to control Proteasomes are giant protein complexes that bind protein molecules and degrade them Chromatin changes Transcription RNA processing mRNA degradation Translation Protein processing and degradation Ubiquitin Protein to be degraded Ubiquinated protein Proteasome and ubiquitin to be recycled Protein fragments (peptides) Protein entering a proteasome Multiple ubiquitin mol- ecules are attached to a protein by enzymes in the cytosol. 1 The ubiquitin-tagged protein is recognized by a proteasome, which unfolds the protein and sequesters it within a central cavity. 2 Enzymatic components of the proteasome cut the protein into small peptides, which can be further degraded by other enzymes in the cytosol. 3

Cancer Biology Mutations are changes in the genetic material of a cell Mutations can occur during DNA replication, recombination, or repair Cancer results from genetic changes that affect cell cycle control Mutation of genes controlling cell division can lead to cancer The gene regulation systems can go wrong due to Chromosomal alterations - translocations Point mutations - Carcinogens Carcinogens are chemical or physical agents that interact with DNA to cause mutations leading to cancer Radiation - X-rays and ultraviolet light Chemicals – arsenic, asbestos, benzene, ethanol, formaldehyde, gasoline Tumor viruses - transform cells into cancer cells through the integration of viral nucleic acid into host cell DNA.

Cancer Cancer results from genetic changes that affect cell cycle control Cancer is the unregulated cell growth and division forming a cluster of cells forming a tumor that constantly expands in size Cells that leave the tumor, spread to other parts of the body, and form new tumors are called metastases

Genes Associated with Cancer The genes that normally regulate cell growth and division during the cell cycle include genes for Growth Factors GF Receptors Intracellular molecules of signaling pathways Mutations altering any of these genes in somatic cells can lead to cancer Most human cancers result from mutations in one of two types of growth-regulating genes: Proto-oncogenes code for proteins involved in stimulating cell division Tumor-suppressor genes code for proteins involved in inhibiting cell division

Growth Factors and Cancer Proto-oncogenes code for proteins involved in stimulating cell division (e.g. growth factors, growth factor receptors, cyclins) Mutated proto-oncogenes that stimulate a cell to divide when it shouldn’t are called oncogenes (cancer-causing genes). Tumor-suppressor genes code for proteins involved in inhibiting cell division Mutated tumor-suppressor genes that do not inhibit cell division when they should can also cause cancer. EFFECTS OF MUTATIONS Protein overexpressed Cell cycle overstimulated Increased cell division Cell cycle not inhibited Protein absent Effects of mutations. Increased cell division, possibly leading to cancer, can result if the cell cycle is overstimulated, as in (a), or not inhibited when it normally would be, as in (b). (c)

Cancer Proto-oncogenes are normal cellular genes that code for proteins that stimulate normal cell growth and division Oncogenes are cancer-causing genes A DNA change that makes a proto-oncogene excessively active, converts it to an oncogene, which may promote excessive cell division and cancer Proto-oncogene DNA Translocation or transposition: gene moved to new locus, under new controls Gene amplification: multiple copies of the gene Point mutation within a control element within the gene Oncogene Normal growth-stimulating protein in excess Hyperactive or degradation- resistant protein New promoter

Ras protein The Ras protein, encoded by the ras gene, is a G protein that relays a signal from a growth factor receptor to a cascade of protein kinases Many ras oncogenes have a mutation that leads to a hyperactive Ras protein that issues signals on its own, resulting in excessive cell division 1 Growth factor (a) Cell cycle–stimulating pathway. This pathway is triggered by a growth factor that binds to its receptor in the plasma membrane. The signal is relayed to a G protein called Ras. Like all G proteins, Ras is active when GTP is bound to it. Ras passes the signal to a series of protein kinases. The last kinase activates a transcription activator that turns on one or more genes for proteins that stimulate the cell cycle. If a mutation makes Ras or any other pathway component abnormally active, excessive cell division and cancer may result. 1 2 4 3 5 GTP Ras Hyperactive Ras protein (product of oncogene) issues signals on its own NUCLEUS Gene expression Protein that stimulates the cell cycle P MUTATION DNA G protein 3 Protein kinases (phosphorylation cascade) 4 2 Receptor Transcription factor (activator) 5

p53 Gene - Tumor-suppressor Gene p53 inhibits cell division when DNA is damaged by stimulating transcription of p21. The p21 protein then binds to cyclins and prevents them from binding with Cdk Abnormal p53 fails to stop division in cells with damaged DNA. If genetic damage accumulates as the cell continues to divide, the cell can turn cancerous. UV light DNA Defective or missing transcription factor, such as p53, cannot activate MUTATION Protein that inhibits the cell cycle pathway, DNA damage is an intracellular signal that is passed via protein kinases and leads to activation of p53. Activated p53 promotes transcription of the gene for a protein that inhibits the cell cycle. The resulting suppression of cell division ensures that the damaged DNA is not replicated. Mutations causing deficiencies in any pathway component can contribute to the development of cancer. (b) Cell cycle–inhibiting pathway. In this 1 3 2 Protein kinases 2 3 Active form of p53 DNA damage in genome 1

CANCER IS CAUSED BY MUTATIONS IN SEVERAL GENES More than one somatic mutation is generally needed to produce a full-fledged cancer cell About a half dozen DNA changes must occur for a cell to become fully cancerous These changes usually include at least one active oncogene and mutation or loss of several tumor-suppressor genes CANCER IS CAUSED BY MUTATIONS IN SEVERAL GENES Tumor suppressor Normal epithelium Hyperpro- liferative Early benign polyp Intermediate benign polyp Late Carcinoma Metastasis APC Oncogene K-ras DCC p53 Other mutations Loss of APC Mutation of K-ras And DCC Mutation of p53

Multistep Model of Cancer Development Colorectal cancer, with 135,000 new cases and 60,000 deaths in the United States each year, illustrates a multistep cancer path Colon Colon wall Normal colon epithelial cells Small benign growth (polyp) Larger benign growth (adenoma) Malignant tumor (carcinoma) 2 Activation of ras oncogene 3 Loss of tumor- suppressor gene DCC 4 Loss of tumor-suppressor gene p53 5 Additional mutations 1 Loss of tumor- gene APC (or other) Colorectal cancer - A multistep model

Inherited Predisposition to Cancer The fact that multiple genetic changes are required to produce a cancer cell helps explain the predispositions to cancer that run in some families Individuals who inherit a mutant oncogene or tumor-suppressor allele have an increased risk of developing certain types of cancer

Cancer Since cancer-causing mutations accumulate over time, cancer risk increases with age.