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Genetics - PCB 3063 • Today’s focus: – Gene Regulation – We will focus on two major questions today: – How do regulatory genes control the activity of multiple genes? – How does gene regulation differ between prokaryotes and eukaryotes? Homework - Due Next Tuesday • The E. coli gal operon is regulated by a repressor (galR) that binds to the galO operator to regulate the structural genes galE, galT, and galK. Expression is induced by the presence of galactose in the medium. If you had cells of the following types, would you expect the structural genes to be inducible, constitutive, or not expressed (give each structural gene) -– – – – galR- galO+ galE+ galT+ galK+ galR+ galOc galE+ galT+ galK+ galR- galO+ galE+ galT+ galK+ / galR+ galO+ galE- galT+ galK+ galR- galOc galE+ galT+ galK- / galR+ galO+ galE- galT+ galK+ • Note: the last two are merodiploids. • Which eukaryotic RNA polymerase transcribes the following genes -– tRNAs – mRNAs – rRNAs First, Let’s Finish Introns... • Typical (canonical) introns follow the GT-AG rule. • Plants and animals also have a small number of non-canonical introns that do not follow the GT-AG (or GU-AG) rule. • There are also tRNA introns removed by specific enzymes. • Self-splicing introns that directly catalyze their own removal have also been found in various genomes. • rRNA is also processed by removal of external and internal transcribed spacers. – However, there is no splicing after ETS and ITS removal, so the ITS and ETS are not introns. RNA Processing - Alternative Splicing • The process of splicing can be controlled - there are introns that are spliced only under specific conditions. • For example, sex determination in Drosophila results from a set of genes with alternatively spliced introns. – E.g., note the skipping of intron 3 in the Sxl (sex lethal) gene of female Drosophila. “Inside-Out” Genes and Inteins • In most genes, the introns are removed and degraded while the EXON sequences are functional. • The snoRNAs are involved in ribosome assembley (small nucleolar RNAs). – One snoRNA (called U22) is present in an intron and the exons are exported to the cytoplasm to be degraded - this is an “inside-out” gene • Tycowski, K. T., Shu, M.-D., and Steitz, J.A. (1996) Nature 379:464-466. • Splicing of proteins has also been described. – These protein introns are called INTEINS. – Inteins are autocatalytically removed from the spliced EXTEIN sequences. – Surprisingly, inteins are related to nucleases. – More information on inteins can be found at: • http://www.neb.com/inteins/intein_intro.html Gene Regulation • There are multiple points at which gene expression is controlled. These include: – Transcription – Translation – Degradation (RNA and/or protein) – Control of protein activity (e.g., by phosphorylation) Nucleus DNA 1Ў transcript (hnRNA) DNA degradation degradation mRNA mRNA Protein Prokaryotic Gene Regulation mRNA Protein Eukaryotic Gene Regulation degradation degradation • Eukaryotes can also control the processing of mRNA and the export of mRNA from the nucleus. – But transcription and translation can interact in prokaryotes. Terminology • A gene that shows increased expression under certain circumstances is said to be INDUCIBLE. – The observation that a gene is induced under certain circumstances does not establish the type of control. – For this reason, one typically discusses changes in the accumulation of mRNAs or proteins. • E.g, an increased amount of mRNA can reflect either transcription (increased), degradation (decreased), or both phenomena. • However, regulation at the level of transcription is fairly common. • Many genes are expressed under a large number of conditions (and in many tissues in complex organisms). These genes are usually called housekeeping genes. – Housekeeping genes are expressed CONSTITUTIVELY. – Karström (1859) concluded that bacterial enzymes may be classified as either constitutive or adaptive. The former are invariably present…the presence of the latter depends on the presence of the substrate upon which they act. The lac Operon • The lac operon is a paradigm for gene regulation. • Regulation of the lac operon occurs at the level of transcription. • Study of the lac operon are based upon a simple observation - E. coli cells synthesize the enzyme b-galactosidase only when two conditions are met: – Lactose is present. – Glucose is absent. A B 1250 1000 Graph of b-galactosidase activity from cells grown in medium with lactose & glucose. A. Glucose exhausted. B. Glucose added back. 750 b-galactosidase activity 500 250 0 0 1 2 3 Time (h) 4 5 b-galactosidase • b-galactosidase catalyzes the cleavage of lactose into glucose and galactose. Glucose + H2O Galactose Lactose • In addition to b-galactosidase, both a lactose permease and b-galactoside transacetylase are induced. • b-galactosidase converts lactose to ALLOLACTOSE, necessary for induction: Allolactose (b-1,6 linkage) The lac Operon - Genes • There are four major genes involved in the lac operon: – – – – lacZ - encodes b-galactosidase lacY - encodes b-galactoside permease lacA - encodes b-galactoside transacetylase lacI - encodes the lac repressor • In addition, one must consider RNAP and CRP (cyclic AMP receptor protein) and several cis acting sites. Trans-Acting Factors & Cis-Acting Sites • There are two major types of transcriptional regulatory entities you should consider: – Trans-Acting Factors - proteins that turn the transcription of genes on or off. – Since they are proteins, they are able to diffuse throughout the cell (hence, they can act in trans - e.g., when they are present in a merozygote) – Cis-Acting Sites - these are typically the binding sites for trans-acting factors. – Since the are specific sites in the DNA that are necessary for gene regulation, they can only act when adjacent to the gene in question. Trans-Ac ting Factor Transc ription Cis-Ac ting Site The lac Operon - Repression • lacI encodes a repressor that binds to the lac operator (lacO), present between the lac promoter (lacP) and the structural genes. – Binding of the lacI gene product to lacO blocks transcription of the polycistronic mRNA encoded by the lac operon. – The structural genes of the lac operon will be regulated in a coordinate fashion, because they are present on the same mRNA. The lac Operon - Derepression • When the lacI encoded repressor binds to inducer (allolactose) it cannot bind the lac operator (lacO). – Thus, transcription of the polycistronic lac operon mRNA is not blocked. – The structural change in the lac repressor caused by allolactose reflects a phenomenon called allosteric regulation. – This model - proposed by F. Jacob & J. Monod - explains induction by lactose (but not regulation by glucose). The Operon Concept • An operon is defined as a unit of co-ordinated gene activity believed to account for the regulation of inducible and repressible enzymes in bacteria (and hence for the regulation of protein synthesis). – The operon is usually conceived as a linear sequence of genetic material comprising an operator, a promoter, and one or more structural genes. • This terminology was introduced by Jacob and Monod in the Journal of Molecular Biology (1961) – Discussing the lac operon they stated that “This genetic unit of co-ordinate expression we shall call the ‘operon’.” – The term REGULON is used if there are multiple operons at distinct locations in the genome that are regulated by the same trans-acting factors. Operon is used if there is a single gene or set of genes at one location with this type of control. Catabolite Repression • The regulation of the lac operon by glucose reflects the phenomenon of catabolite repression. – CRP (cyclic AMP [cAMP] receptor protein), also called CAP (catabolite activator protein) binds to a cis-acting site and promotes RNAP binding. • CRP promotes RNAP binding by bending DNA. – CRP binds DNA when it binds to cAMP. • cAMP is produced when glucose is absent. cAMP and Catabolite Repression • cyclic AMP (cAMP) accumulates when glucose is absent. – Adenylcyclase is inhibited by glucose. – cAMP phosphodiesterase is activated by glucose. Adenyl cyclase cAMP ATP Phosphodiesterase AMP The lac Operon: Multiple Signals • These two regulatory systems result in the observed regulatory pattern: Lactose Glucose cAMP lac Operon + + + + - Low No mRNA High Little mRNA Low No mRNA High High mRNA accumulation – In addition to these patterns, it was also noted that some mutations in lac genes abolished the activity of genes that follow them in the operon. • e.g., there are lacZ mutants that do not express lacY or lacA. – These are called POLAR MUTATIONS. – These mutations are NONSENSE MUTATIONS. • The phenotype of polar mutations reflects problems with the translation of downstream cistrons when there are mutations in the upstream genes. The trp Operon - Repression • Tryptophan biosynthetic genes are also regulated, but the mechanism is quite different: – In this case, tryptophan (trp) binds to a repressor (the product of trpR) that works in a manner similar to the lac repressor. – However, the binding of Trp to the trpR gene product causes it to bind DNA. – Thus, Trp is called a COREPRESSOR in this context. The trp Operon - Attenuation • However, trp operon regulation is more complex: – C. Yanofsky established that the leader sequence of the polycistronic transcript of the trp operon contains a short open reading frame (ORF) with a ribosome binding site (RBS). – This ORF has two tryptophan codons, which can cause a ribosome to stall during translation if it is limiting. Attenuation - Stem Loops • The trp operon mRNA leader can adopt several structures: • If the ribosome stalls, it causes a structure that does not result in termination. • Either read-through or the absence of translation allow the mRNA to adopt a structure that results in termination. – Could this mechanism of gene regulation occur in eukaryotes? Attenuation - Variation • In E. coli and Salmonella, there are other amino acid biosynthetic operons regulated by attenuation: Regulatory Amino Operon Leader Acids MTRVQFKHHHHHHHPD His His Phe MKHIPFFFAFFFTFP Phe Leu MSHIVRFTGLLLLNAFIVR... Leu MKRISTTITTTIT ITTGNGAG Thr Thr, Ile MTALLRVISLVVISVVV IIIP... Ilv Ile, Val • In B. subtilis, the trp operon mRNA is also controlled by attenuation. – However, it does not involve ribosome binding. – Instead, there is a protein called TRAP (trp RNA binding attenuation protein) that binds the leader of the trp mRNA. – TRAP binds Trp (11 molecules) and then binds the leader, causing attenuation. GAL Genes: Eukaryotic Transcriptional Regulation • Unlike prokaryotes, eukaryotes do not have genes in operons (most mRNAs are not polycistronic). • The GAL genes of S. cerevisiae are the paradigm for eukaryotic gene regulation • Galactose is metabolized by GAL gene products: Galactose Gal1p UDP-Glu Gal10p Gal-1-P Gal7p UDP-Gal Glu-6-P Glycolysis Gal5p Glu-1-P Eukaryotic Transcription • Proteins bind to distal elements called ENHANCERS. • DNA folding allows these elements to be far from the start site for transcription. • Proteins bound to the distal sites promote the binding of RNA polymerase to the proximal elements. Distal Proximal GAL Genes: A Transcriptional Program • The response to galactose is very complex, with a number of genes being turned on or off. • The central regulator is a protein called Gal4p. – Gal4p binds to enhancer elements in DNA and activates transcription under some circumstances. Gal4p: A Transcriptional Regulator • Gal4p binds to enhancer elements near genes that it regulates (e.g., GAL1). • Gal4p also binds to Gal80p. – Gal80p is necessary for activation of gene expression. • When galactose binds to Gal80p, the Gal4p-Gal80p complex can activate transcription. – This activation has now been studied at the level of the whole genome: • This figure shows data from a microarray experiment (Science 290:2306 [2000]). Examining Transcriptional Regulation • MICROARRAYS have become very popular as tools to study gene regulation. – A microarray is a small glass slide on which cDNAs of many (or all) genes in an organism have been dotted. – cDNA is made using mRNAs present under certain conditions (or in a certain tissue) and labeled with fluorescent dyes. – Then, the labeled cDNA are hybridized to the microarray and the fluorescence determined. • There is a nice animation describing this at: – http://www.bio.davidson.edu/courses/genomics/chip/chip.html – Does this examine transcriptional regulation? Examining Transcriptional Regulation • This basic method was extended for the Gal4p study that we have been discussing discussed. – For this study, the researchers tagged the Gal4p protein so the could purify from the cell. – Then, they chemically cross-linked it to DNA and purified it. – This allowed them to purify the DNA that Gal4p was bound to in the cell. – The DNA that Gal4p was bound to in the cell was labeled and used to probe the microarray. – Does this examine transcriptional regulation? Examining Transcriptional Regulation • This study established several interesting facts: – The Gal4p binding sites in the DNA are sometimes bound by Gal4p in the absence of galactose, others are bound only in the presence of galactose. – So the trigger is more complex than simply whether or not the Gal4p protein can bind. – This more complex regulation involves Gal80p, an inhibitor. Two possible models for regulation of the Gal4p-Gal80p complex by galactose. The models differ only in the exact binding sites for Gal80p. How do Eukaryotic Transcriptional Regulators Work? • There are a few specific types of proteins that act to increase transcriptional activity: – Many proteins have an acidic domain. • Surprisingly, these “acid-blob” proteins often require a hydrophobic residue embedded in an acidic region. • Both Gal4p and the herpes simplex virus VP16 protein (an transcriptional regulator for this virus) have acid blobs. – Glutamine-rich and Proline-rich transcriptional activation domains have been characterized. • These protein regions activate transcription when fused to other DNA-binding domains. – Alternatively, they can be recruited by protein-protein interactions - e.g., a DNA-binding protein binds the enhancer, and it contains a region that recruits and acid-blob protein. Using Eukaryotic Transcriptional Regulators • The yeast 2-hybrid system exploits these features of eukaryotic transcription factors to examine proteinprotein interactions. – The DNA-binding and transcription activating regions of Gal4p can be separated. – Interestingly, if you fuse one protein to the Gal4p DNA-binding domain (BD) and a second protein that it interacts (physically) with to the Gal4p transcriptional activating domain (AD), one can see transcriptional activation: How do Eukaryotic Transcriptional Regulators Work? • Another interesting phenomenon that is sometimes seen with transcription factor is SQUELCHING. – Overexpression of transcription activators like Gal4p can result in a general inhibition of transcriptional activity. – How does this happen? – Presumably, specific transcription factors like Gal4p act by recruiting “basal” transcription factors. • In fact, some basal factors that physically interact with these transcription activating domains have been found. • Basal factors are factors involved in recruiting RNA polymerase II to a large number of promoters. – So overexpressing proteins with these transcription activating domains can actually turn gene expression off, by competing for these factors. How do Eukaryotic Transcriptional Regulators Work? • At least one way is by altering the packing of DNA into chromatin. • The role of chromatin structure in the regulation of transcription is an area of very active investigation. • However, two important factors that play clear roles in transcriptional regulation are known: – DNA METHYLATION - A subset of cytosine (C) residues are modified by methylation. – HISTONE ACETYLATION - Histones can be modified by acetylation. Chromatin • Remember, DNA in eukaryotes packs into CHROMATIN. • HISTONES form the NUCLEOSOME, which DNA loops around. • EUCHROMATIN - less compact; actively transcribed • HETEROCHROMATIN more compact; transcriptionally inactive. – Heterochromatin can be either constitutive or facultative. DNA Methylation • Genes that are transcriptionally inactive are often METHYLATED. – In eukaryotes, cytosine residues are modified by methylation. NH2 NH2 CYTOSINE CH3 N O N N H O N H METHYL-C • Typically, the sites of methylation are CG dinucleotides (vertebrates). – This allows maintenance through replication. Histone Acetylation • HISTONES in transcriptionally active genes are often ACETYLATED. • Acetylation is the modification of lysine residues in histones. – Reduces positive charge, weakens the interaction with DNA. – Makes DNA more accessible to RNA polymerase II • Enzymes that ACETYLATE HISTONES are recruited to actively transcribed genes. • Enzymes that remove acetyl groups from histones are recruited to methylated DNA. – There are additional types of histone modification as well, such as methylation of the histones. Genetic Imprinting • Remember that DNA methylation can be maintained through replication. • This allows the packing of chromatin to be passed on just like a gene sequence. – However, differences in chromatin packing are not as stable as gene sequences. • Heritable but potentially reversible changes in gene expression are called EPIGENETIC phenomena – Vertebrates use these differences in chromatin packing to IMPRINT certain patterns of gene regulation. – Some genes show MATERNAL IMPRINTING while other show PATERNAL IMPRINTING. • The alleles of some genes that are inherited from the relevant parent are methylated, and therefore are not expressed. Prader-Willi & Angelman Syndromes • Both of these genetic disorders are caused by deletion of a region of chromosome 15. • However, the syndromes differ: – Prader-Willi Syndrome - obesity, mental retardation, short stature. (abbreviated PWS) – Angelman Syndrome - uncontrollable laughter, jerky movements, and other motor and mental symptoms. (abbreviated AS) • Syndrome that develops depends upon the parent that provided the mutant chromosome. PWS AS PWS Mouse model AS Mouse model From Annu Rev Genomics & Hum Genet Prader-Willi & Angelman Syndromes • Prader-Willi Syndrome - develops when the abnormal copy of chromosome 15 is inherited from the father. • Angelman Syndrome - develops when the abnormal copy of chromosome 15 is inherited from the mother. • The differences reflect the fact that some loci are IMPRINTED - so only the allele inherited from one parent is expressed. – The region contains both maternally and paternally imprinted genes. Methylation and Gene Regulation • For imprinted genes, the pattern of gene regulation is dependent upon the parent that donated the chromosome. – The methylation pattern is “reprogrammed” in the germ line. • There are other examples of methylation changes the regulate gene expression. – In mammals, one of the two X chromosomes in females is inactivated. – The inactivated X is methylated.