Transcription-regulation

Transcription-regulation The lac operon 242 Metabolism of lactose In the 1950s, pioneering experiments were carried out by François Jacob and Jacques Monod who studied regulation of gene transcription in E. coli by analyzing the expression of enzymes involved in the metabolism of lactose Components of the lac operon Lactose induces the synthesis of enzymes involved in its own metabolism including: -galactosidase: catalyzes the cleavage of lactose lactose permease: transports lactose into the cell a transacetylase: acetylates -galactosides These genes are located in one operon known as the lac operon 244 What is an operon? A cluster of genes transcribed from one promoter producing a polycistronic mRNA 245 The operator The DNA region that regulates gene expression (transcription) is called a promoter. Usually this region is localized right before the start site of transcription It includes the RNA polymerase binding site The promoter also includes a region known as the operator region that also regulates transcription 246 The i protein (lac repressor) Transcription of the lac operon is also controlled by a protein expressed by the i gene The i protein (lac repressor) blocks transcription by binding to the operator preventing the RNA polymerase from biding to the promoter 247 Regulation by lactose (positive) The addition of lactose leads to induction of the operon because lactose binds to the repressor, thereby preventing it from binding to the operator DNA This is known as positive regulation 248 249 Cis vs. trans regulatory elements Regulatory sequences like the operator are called cisacting control elements, because they affect the expression of only linked genes on the same DNA molecule Proteins like the repressor are called transacting factors because they can affect the expression of genes located on other chromosomes within the cell 250 Effect of mutations Mutations affecting o result in constitutive expression (always on) since these mutations prevent i from binding to the operator Mutants of i are either constitutive or noninducible (always off) In constitutive i mutants, i always binds lactose, so expression of the operon is always induced In noninducible i mutants, the repressor binds to the operator very tightly even in the presence of lactose 251 Regulation by glucose (negative) Glucose is preferentially utilized by bacterial cells If E. coli are grown in medium containing both glucose and lactose, the lac operon is not induced and only glucose is used by the bacteria Glucose represses the lac operon even in the presence of the normal inducer (lactose) This is known as negative regulation 252 How does glucose repress the expression of the lac operon? Low glucose activates the enzyme adenylyl cyclase, which converts ATP to cAMP cAMP then binds to catabolite activator protein (CAP) cAMP stimulates the binding of CAP to DNA upstream of the promoter CAP then interacts with the RNA polymerase, facilitating the binding of polymerase to the promoter and activating transcription 253 254 Positive vs. negative regulation 256 257 Regulatory mechanisms Although the control of gene expression is far more complex in eukaryotes than in bacteria, the same basic principles apply Transcription in eukaryotic cells is controlled by: Cis-acting DNA sequences Transcriptional regulatory proteins Repressor proteins Modification of DNA and its packaging into chromatin 258 Regulatory sequences Promoters and enhancers As already discussed, transcription in bacteria is regulated by the binding of proteins to cis-acting sequences (e.g., the lac operator) Similar cis-acting sequences regulate the expression of eukaryotic genes: promoters enhancers 259 General components of promoters An upstream elements, which act as enhancers and silencers. The TATA box, which has a consensus sequence of TATAA(T/A). The initiator element (Inr), which surrounds the +1 site. A possible downstream regulator 260 Enhancers Genes that are regulated solely by the consensus elements in the promoter region are constitutively expressed. Gene-specific transcription factors (also called transactivators or activators) are located farther away from the transcription start site and bind to gene-specific regulatory sequences and interact with mediator proteins such as coactivators. These cis-acting regulatory sequences, also called enhancers, function by binding transcription factors that then regulate RNA polymerase. They can stimulate transcription when placed either upstream or downstream of the promoter, in either a forward or backward orientation because of DNA looping 261 262 Transcriptional regulatory proteins These proteins to consist of two domains: One region of the protein specifically binds DNA (DNAbinding domain) the other activates transcription by interacting with other components of the transcriptional machinery (regulatory or activation domain) Both activities are independent and can be separated from each other 263 DNA-binding domains Zinc finger domains (Steroid receptors) helix-turn-helix motif leucine zipper (CREB) helix-loop-helix 264 The activation domains These activation domains are thought to stimulate transcription by interacting with general transcription factors, such as TFIIB or TFIID, thereby facilitating the assembly of a transcription complex on the promoter Acidic domains Glutamine-rich domains Proline-rich domains 265 cAMP-response element (CRE) • Unphosphorylated CREB does not bind to CREB binding protein, and no transcription occurs. • In the presence of cAMP, protein kinase A is activated phosphorylating CREB, which binds to CBP. The dimer can then form a complex with the RNA polymerase and, thereby, activating transcription. 266 Huntington’s disease It is a disease caused by a mutation in a transcription protein known as huntingtin. The mutation is an increase in the number of a trinucleotide repeat of CAG (it encodes polyglutamine). The polyglutamine product sequesters CBP, making less of it available for molecular processes, such as transcription and DNA repair. The loss of the DNA repair leads to a propagation of the CAG repeats and leads to the disease becoming worse in successive generations. Eukaryotic Repressors Repressors bind to specific DNA sequences and inhibit transcription Repressors may have both DNA-binding and protein-binding domains DNA-binding domains, but not protein-interaction domains protein-interacting domains, but not DNA-binding domains 268 Steroid receptors The receptors to which lipophilic steroid hormones bind are ligand-activated proteins that regulate transcription of selected genes They are found in the cytosol and the nucleus Upon hormonal binding, the hormone-receptor complex bind to specific DNA promoter/enhancer sequences 269 Trancriptional regulatory network (primary, secondary,…etc transcription regulation 270 Epigenomics Epi: “above” or “in addition to” It indicates genetic alterations in gene expression without a change in DNA sequence Can be caused by the pattern of chromosomal packaging and modification (e.g. methylation) 271 Modulation of chromosomal structure The packaging of eukaryotic DNA in chromatin has important consequences in terms of its availability as a template for transcription Actively transcribed genes are found in loose chromatin, and vice versa Even in loose chromatin, the tight winding of DNA around the nucleosome core particle can prevent transcription factors from binding DNA and the RNA polymerase to transcribe through a chromatin template 272 273 274 275 How are chromosomal structures altered? Decrease in the compactness of the chromatin by: Acetylation of histones Binding of specialized nonhistone chromosomal proteins to nucleosomes of actively transcribed genes to change their conformation. Complexing of transcription factors with nucleosome remodeling factors. 276 Histone acetylation 277 Enzymatic association Transcriptional activators and repressors are associated with histone acetyltransferases and deacetylases, respectively A component of TFIID has been found to be a histone acetyltransferases 278 Nucleosome remodeling factors These are protein complexes that facilitate the binding of transcription factors by altering nucleosome structure by the accessibility of nucleosomal DNA to transcription factors and other proteins without removing the histones 279 DNA Methylation Cytosine residues in DNA can be modified by the addition of methyl groups at the 5-carbon position DNA methylation is correlated with reduced transcriptional activity of genes 280 Mechanism of inhibition Methylation inhibits transcription of genes via the action of a protein, MeCP2, that specifically binds to methylated DNA and represses transcription Interestingly, MeCP2 functions as a complex with histone deacetylase, linking DNA methylation to histone acetylation and nucleosome structure 281 Significance of DNA methylation DNA methylation has been established in two important phenomena: X chromosome inactivation In females, one of the X chromosomes is inactivated in every cells. genomic imprinting Either the paternal gene or the maternal gene is active. 282 How do different cells appear? All cells in one body have the same genome, but cell types in a multicellular organism become different from one another because they synthesize different sets of RNA and protein molecules. The patterns of mRNA can differentiate cell types from each other. The transcriptome (the total collection of RNA transcripts in a cell) 283 Northern blotting This is done exactly like Southern blotting except that RNA from cells is isolated instead of DNA. Then RNA molecules are fractionated based on sizes by gel electrophoresis. The fractionated RNA molecules are transferred to a membrane. The RNA molecules on the membrane are targeted by a labeled DNA probe whose sequence is complementary to a specific RNA molecule. 284 285 286 In situ hybridization In situ hybridization methods reveals the distribution of specific RNA molecules in cells in tissues. RNA molecules can hybridize when the tissue is incubated with a complementary DNA or RNA probe. In this way the patterns of differential gene expression can be observed in tissues, and the location of specific RNAs can be determined in cells. 287 RNA expression in drosophila 288 289 290 What is a DNA library? A library can be created for DNA fragments just like book libraries. You can have clones of bacteria each containing a specific piece of DNA. You can save these clones in the freezer and take whichever clone you want to study. 291 Genomic DNA library 292 cDNA library This library contains only those DNA sequences that are transcribed into mRNA. This is done by extracting the mRNA from cells and then making a complementary DNA (cDNA) copy of each mRNA molecule present. cDNA is made by retroviral reverse transcriptase. 293 Genomic vs. cDNA libraries Genomic clones represent a random sample of all of the DNA sequences in an organism. By contrast, cDNA clones contain only those regions of the genome that have been transcribed into mRNA. Because the cells of different tissues produce distinct sets of mRNA molecules, a distinct cDNA library is obtained for each type of cell used to prepare the library. 294 RT-PCR of mRNA Another way of relative quantitation of RNA expression is by converting RNA into cDNA followed by PCR in the presence of SYBR green. The higher the amount of RNA (cDNA), the sooner it is detected. The science of -omics 296 Studying the transcriptome One such method in studying transcriptomes is DNA microarrays, which allow the analysis of the RNA products of thousands of genes all at once. By examining the expression of so many genes simultaneously, we can understand gene expression patterns in physiological and pathological states. 297 DNA microarrays DNA microarrays are glass microscope slides spotted with up to tens of thousands of DNA fragments in an area the size of a fingernail. The exact sequence and position of every DNA fragment on the array is known. 298 299 A DNA microarray This is done for a single sample using radioactively labeled cDNA. mRNA from the cells being studied is first extracted and converted to cDNA. The cDNA is labeled with a radioactive probe. The microarray is incubated with the labeled cDNA sample for hybridization to occur. If a gene is expressed, then the cDNA will exist and bind to a specific complementary DNA fragment on the microarray. Binding can be detected since the cDNA is labeled and expression is determined. 300 Comparative expression In order to compare expression of genes two different samples, the cDNA molecules are fluorescently labeled with different colors (green and red) and added to the array. An increases in the amount of a RNA molecule in one sample versus the other is reflected by an increase the amount of produced cDNA and an increase in fluorescence in the bound spot. 301 302 Sample 1 Sample 2 Sample 3 1 2 3 1 2 3 1 2 3 4 5 6 4 5 6 4 5 6 7 8 9 7 8 9 7 8 9 Combine results Eliminate samples 2, 4,& 9 Cluster samples according to expression 1 5 Genes 3 8 7 6 Samples 1 3 2 This can be done at a larger scale whereby samples are compared to the same control sample and a computer program combines all data illustrating differences in expression among the samples and classifying them into different groups. 303 DNA microarrays and breast cancer 304