Gene Expression 1 The process by which a gene's information is converted into the structures and functions of a cell by a process of producing a biologically functional molecule of either protein or RNA (gene product) is made. Gene expression is assumed to be controlled at various points in the sequence leading to protein synthesis. 2 Every cell has the same DNA and therefore the same genes. But different genes need to be on and off in different types of cells. Therefore, gene expression must be regulated. embryo bone liver muscle sperm (The first statement on this slide is not completely true. Which of these cells does not have exactly the same DNA as the others?) Gene expression must be regulated in several different dimensions 10 wks14 wks 1 day 6 mos 12 mos18 mos In time: At different stages of the life cycle, different genes need to be on and off. M. Halfon, 2007 In space: Paddock S.W. (2001). BioTechniques 30: Each colored stripe in this fly embryo shows the expression of a different gene or set of genes. The spatial regulation of these genes allows the embryo to be divided up into different regions that will give rise to the head, the internal organs, the abdomen, etc. and in abundance: Clyde et al. (2003). Nature 426: Note how the gene whose expression is indicated in blue varies in abundance from strong expression (bold arrow) to weak (thin arrow) within its expression domain. These differences in strength of gene expression have important functional consequences. behavior pattern chromosome inactivation metabolism pathology (mutation) common variation evolution Importance of gene regulation M. Halfon, 2006 Gene Regulation and Nutrition: Metabolism (enzymes) high carb/low fat (sustained) insulin increased transcription acetyl-CoA carboxylase fatty acid synthase Your long-term diet can lead to permanent changes in your bodys gene expression profile Gene Regulation and Nutrition: Development (organs, cell types) embryo muscle brain embryo intestines fat liver (diseased) With respect to nutrition, gene regulation is important to guide the development of organs, tissues, and cell types required to ingest, digest, and metabolize nutrients. Gene Structure Eukaryotic gene structure: Most eukaryotic genes in contrast to typical bacterial genes, the coding sequences (exons) are interrupted by non-coding DNA (introns). The gene must have exons; start signals; stop signals; regulatory control elements. The average gene is 7-10 exons spread over 10-16kb (kilo base pairs) of DNA. 10 Genes can be regulated at many levels Central Dogma of Biology Control of Gene ExpressionTranscription Factors Transcription factors (TFs) are proteins that bind to the DNA and help to control gene expression. We call the sequences to which they bind transcription factor binding sites (TFBSs). Control of Gene Expression: Promoters Every gene has a promoter, the DNA sequence immediately surrounding the transcription start site. The promoter is the site where RNA polymerase and the so- called general transcription factors bind. Control of Gene Expression Image adapted from Wolpert, Principles of Development Transcription factor binding sites are found within larger functional units of the DNA called cis-regulatory elements. There are two main type of cis-regulatory elements: promoters, and cis-regulatory modules (sometimes called enhancers). TFBS transcription factor binding site (TFBS) cis-regulatory module (CRM) Control of Gene Expression: Promoters Every gene has a promoter, the DNA sequence immediately surrounding the transcription start site. The promoter is the site where RNA polymerase and the so- called general transcription factors bind. Additional gene regulation takes place via the cis- regulatory modules (CRMs), which can be located 5 to, 3 to, or within introns of a gene. CRMs can be very far away from the gene they regulateover 50 kband other genes might even lie in between! Control of Gene Expression: CRMs TFBS transcription factor binding site (TFBS) cis-regulatory module (CRM) TFBS transcription factor binding site (TFBS) cis-regulatory module (CRM) Looking at cis-regulatory modules: Reporter Genes green fluorescent protein minimal promoter CRM muscle e.g., from Myosin Heavy Chain gene Looking at cis-regulatory modules: Reporter Genes A reporter gene assay is used to identify CRMs by recombinant DNA methods The jellyfish green fluorescent protein (GFP) gene is often used, as the encoded protein emits green light when exposed to light of the proper wavelength. We can test for CRM activity in transfected cells in culture, or even better, in a transgenic animal : 19 Prokaryotic vs. Eukaryotic Bacterial genetics are different. Prokaryote genes are grouped in operons. Prokaryotes have one type of RNA polymerase for all types of RNA, mRNA is not modified The existence of introns in prokaryotes is extremely rare. Operons- How Bacteria regulate An operon is a group of genes that are transcribed at the same time. They usually control an important biochemical process. They are only found in prokaryotes. NobelPrize.org Jacob, Monod & Lwoff 2007 Paul Billiet ODWSODWS 1. When lactose is absent A repressor protein is continuously synthesised. It sits on a sequence of DNA just in front of the lac operon, the Operator site The repressor protein blocks the Promoter site where the RNA polymerase settles before it starts transcribing Regulator gene lac operon Operator site zya DNA I O Repressor protein RNA polymerase Blocked 2007 Paul Billiet ODWSODWS 2. When lactose is present A small amount of a sugar allolactose is formed within the bacterial cell. This fits onto the repressor protein at another active site (allosteric site) This causes the repressor protein to change its shape (a conformational change). It can no longer sit on the operator site. RNA polymerase can now reach its promoter site zya DNA IO 2007 Paul Billiet ODWSODWS 2. When lactose is present A small amount of a sugar allolactose is formed within the bacterial cell. This fits onto the repressor protein at another active site (allosteric site) This causes the repressor protein to change its shape (a conformational change). It can no longer sit on the operator site. RNA polymerase can now reach its promoter site Promotor site zya DNA I O 2007 Paul Billiet ODWSODWS Many adult humans cannot metabolize lactose (milk sugar). A single nucleotide polymorphism (SNP), i.e., a one base pair difference in DNA sequence, correlates with activation of the lactase promoter and with lactose tolerance/intolerance. Moreover, this simple change can be seen to affect the binding activity of a transcription factor, Oct-1, to the relevant CRM. There are likely to be many such instances of how changes in gene regulation affect nutrition, health, and disease, although most remain to be discovered. Olds, L. C. and E. Sibley (2003). Hum. Mol. Genet. 12(18): agataatgtagTccctggcctca agataatgtagCccctggcctca tolerant intolerant ++ + Oct-1 binding phenotype ++ + ability to activate A nutritional example: the lactase gene M. Halfon, 2007 Control of Gene Expression 26 27 The human genome contains about 20,325 genes - However, these encode about 100,000 mRNAs, which in turn specify more than a million proteins Several events account for the fact that proteins outnumber genes Maximizing Genetic Information The genes in pieces pattern of exons and introns and alternate splicing help to greatly expand the gene number Maximizing Genetic Information Figure 11.9 Only 1.5% of human DNA encodes protein Rest of genome includes: - Viral DNA - Noncoding RNAs - Introns - Promoters and other control sequences - Repeated sequences Most of the Human Genome Does Not Encode Protein About 8% of our genome is derived from RNA viruses called retroviruses - This is evidence of past infection - Sequences tend to increase over time Viral RNA Figure 11.11 Nearly all of the human genome can be transcribed, and much of it is in the form of noncoding RNAs (ncRNAs) This includes rRNAs and tRNAs However, there are hundreds of thousands of other ncRNAs - These are transcribed from pseudogenes - But are not translated into protein Noncoding RNAs MicroRNAs belong to a class of molecules called noncoding RNAs They are bases long The human genome has about 1,000 distinct microRNAs that regulate at least 1/3 rd of the protein-encoding genes When a microRNA binds to a target mRNA, it prevents translation MicroRNAs Cancer provides a practical application of microRNAs because certain microRNAs are more or less abundant in cancer cells than in healthy ones A related technology is called RNA interference (RNAi) - Small synthetic, double-stranded RNA molecules are introduced into selected cells to block gene expression MicroRNAs Transposons are the most abundant type of repeat - Sequences that jump about the genome - All repeats can copy themselves - Comprise about 2-3% of the genome Rarer classes of repeats include those that comprise telomeres, centromeres, and rRNA gene clusters Repeats Transposon Animation Please note that due to differing operating systems, some animations will not appear until the presentation is viewed in Presentation Mode (Slide Show view). 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