Lecture Notes for Exam 4
Lecture Notes for Exam 4 BIOL 5344
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Transcriptional Regulation in Bacteria (I):▯ General Principles▯ and the lac Operon Monday, November 2, 2015 BIOL 5304 Lecture 26 The ﬁnal topic in this course is Regulation So far we have looked at: Copying, recombining, and repairing of DNA Transcription of DNA into RNA Processing of RNA into mature mRNA Charging of tRNA Translation of mRNA into polypeptides Many of these processes are quite similar in a wide variety of organisms, from common bacteria to animals. Gene regulation in prokaryotes is primarily at the level of transcriptional initiation. 1. It is energetically efﬁcient to regulate an early step 2. Mechanistically, it is more efﬁcient to regulate a single gene, rather than hundreds of mRNAs. Why is regulation not exclusively at transcriptional initiation? 1. If there are multiple points of regulation, there can be more paths of modulation, i.e. more signals can have input. 2. Regulation at the translational level allows a faster response for expression. Transcriptional Regulation in Bacteria Remember that transcription requires an RNA polymerase and a sigma factor to bind at a promoter. The RNA polymerase must transform into an open complex, with a transcription bubble, and release the sigma factor, before transcription is fully underway. This yields the basal level of transcription. In addition, both positive regulators, activators, and negative regulators, repressors, can inﬂuence the rate of transcription. Some regulators work Fig. 18-1 simply at the level of binding.▯ In the absence of other factors there will be a basal rate of transcription.▯ If the RNA polymerase is blocked from binding,then there will be no transcription.▯ If binding of the RNA polymerase is enhanced, then the rate of transcription will increase (be activated). 5 The binding of the Fig. 18-1 RNA polymerase can be blocked by a repressor, which binds to an operator. If the promoter is weak, or is lacking UP elements, the binding of the RNA polymerase can be enhanced by simultaneous binding to an activator at a nearby site. Now it is held until it assumes the open complex. This is an example of cooperativity. 6 Another kind of promoter does not allow the open complex to form until a conformational change in the RNA polymerase occurs. In this case, the role of the activator is to cause the conformational change. This is an example of allostery, in which the binding of one protein (the activator) changes the conformation of another (the RNA polymerase). Fig. 18-2 Activator sites do not need to be adjacent, as shown in (a), due to the looping ability of DNA, as shown in (b). In bacteria, these distances can be several kilobases. Fig. 18-3 Sometimes, special DNA-bending proteins are involved in allowing activators to act at a distance. Fig. 18-4 Anti-terminators Regulation of bacterial transcription is not restricted to the events of initiation. In a later lecture we will learn about anti-termination, in which transcription normally is terminated (prematurely), unless something intervenes and allows it to ﬁnish. There are also other control methods involving small RNA molecules, and RNA structure. Cooperativity of binding can be extended to multiple partners, allowing an integration of signals to determine a rate of transcription. The conformation of activators and repressors, and hence their ability to function, can be strongly inﬂuenced by the binding of small molecules, as we will see in the example considered today: the lac operon There are 3 genes in the lac operon: lacZ, coding for β-galactosidase lacY, coding for lactose permease lac A, coding for thiogalactoside transacetylase Fig. 18-5 β-galactosidase: cleaves lactose into galactose and glucose lactose permease: A membrane protein that transports lactose into the cell. It is a symporter of H and lactose. thiogalactoside transacetylase: An enzyme that modiﬁes the toxic thiogalactosides that are also transported by lactose permease. It is important for detoxiﬁcation. There is one promoter for these 3 genes. It is adjacent to an upstream activator binding site (CAP site), and it overlaps with a repressor binding site (operator). Fig. 18-5 CAP is the Catabolite Activator Protein, also called CRP , cyclic AMP Receptor Protein. It mediates the effect of glucose. It is a DNA-binding protein. The repressor is the Lac repressor, product of lacI. It mediates the effect of lactose. It is also a DNA-binding protein. Fig. 18-6 In the absence of lactose, the lac sugars in the repressor binds, growth and blocks the medium work? RNA polymerase. There is no need for these genes to be expressed. Since glucose is the preferred carbon source, in the presence of glucose, there is no activation through the CAP site. How does the lac repressor work? It binds to a symmetrical DNA sequence, suggesting that it is also 2-fold symmetrical. 8 of the 10 base pairs in each half are identical the circled base pairs are not. Fig. 18-7 The -10 promoter site is adjacent to the operator, but since the RNA polymerase is rather large, it cannot bind to the promoter if the repressor is bound to the operator. Fig. 18-8 In contrast, the CAP site can be occupied by CAP while the RNA polymerase is bound to the promoter. CAP provides favorable interactions for binding the RNA polymerase. The CAP binding site is also symmetrical. Without CAP , the RNA polymerase binds rather weakly because its -35 region is not optimal. Also, there is no UP-element. These binding sites were originally mapped by DNA footprinting techniques. DNA foot-printing experiments can show that proteins bind to speciﬁc regions of DNA. Box 18-1 Fig. 1 On the left, the On the right, the DNA is subjected DNA with a to random binding protein, is digestions, subjected to resulting in a random digestions. ladder of In this case, the fragments of protein protects different sizes, the DNA from corresponding to digestion, in a the sites of speciﬁc region. digestion. Schematic view of how CAP interacts with the C-terminal domain of the alpha subunit of the RNA polymerase. Fig. 18-9 CAP activates by simple recruitment of the RNA polymerase. Mutagenesis experiments showed that regions of CAP interact with the CTD (carboxy terminal domain) of the alpha subunit. CAP binds as a dimer, each half requiring a molecule of cAMP . The CTD of the alpha subunit of the RNA Polymerase is shown in magenta, binding to DNA, and to one-half of the CAP . cAMP analog Structural tutorial Fig. 18-10 Both CAP and the lac repressor bind to the DNA using the helix-turn-helix motif. One alpha helix, labeled R binds along the major groove of the DNA. Fig. 18-11 Illustration of the interactions between a helix-turn-helix (2 and 3) and the bases in the major groove of DNA. Figure from a previous edition Here is CAP , bending the DNA about 90˚. The helix-turn-helix motifs are in blue. Each monomer of CAP binds a molecule of cyclic AMP , which is necessary for DNA binding. cAMP is an effector molecule The C-terminal domains of the α-subunits are shown in green and magenta. The HTH motifs of CAP are in blue, and a second motif that binds to DNA is in red. The magenta α-CTDs contact both CAP and DNA. The green α-CTDs contact DNA only Structural basis of transcription activation: the CAP-alpha CTD-DNA complex. Benoff B, Yang H, Lawson CL, Parkinson G, Liu J, Blatter E, Ebright YW, Berman HM, Ebright RH. Science. 2002 Aug 30;297(5586):1562-6. 23 Three different conformations of CAP have been captured in crystal form. The binding of cyclic AMP causes the central helices to extend. This turns the golden helices by about 90 degrees, so that they ﬁt into the major grooves. DBD: DNA-Binding Domain CBD: Cyclic-AMP Binding Domain Fig. 18-14 The lac repressor actually functions as a tetramer. There are 2 additional operators nearby. One is upstream (90 bp) and one is downstream (400 bp) Fig. 18-12 The additional binding sites increase the afﬁnity of repressor for the operator. Some current research is addressing the effect of supercoiling on the ability of lac repressor to bind looped DNA. Each lac operator is bound by two subunits of lac repressor. This shows the DNA-binding domains of 2 repressor subunits bound to a single operator sequence. The helix-turn-helix (HTH) motifs are colored blue. These motifs bind in major grooves, as is normal. The extra helix (red) binds in the minor groove, at the center of the operator, as a pair. This is unusual for a repressor. Plasticity in protein-DNA recognition: lac repressor interacts with its natural operator 01 through alternative conformations of its DNA-binding domain. Kalodimos CG, Bonvin AM, Salinas RK, Wechselberger R, Boelens R, Kaptein R. EMBO J. 2002 Jun 26 17;21(12):2866-76. The binding of lactose (actually its derivative allolactose) changes the conformation of the repressor, preventing it from binding to the operator. Allolactose is the inducer of the lac operon. It is produced from lactose by β-galactosidase. IPTG is an artiﬁcial Fig. 18-13 inducer of the lac X-gal is a substrate of β-galactosidase that operon, used in lab work. yields a colored product upon hydrolysis. isopropyl β-D-1- 4-chloro-3-indolyl β-D-galactopyranoside thiogalactopyranoside The binding of lactose (actually its derivative allolactose) changes the conformation of the repressor, preventing it from binding to the operator. The inducer binding site is shown in green, occupied by an anti-inducer. Allolactose, or IPTG, are effector molecules that changes the conformation of the dimeric lac repressor. An anti-inducer binds to the same site as an inducer, but it has the analysis of Lacc opposite effect. repressor bound to natural operator O1.re the complex J Mol Biol. 2001 Oct held together. 28 5;312(5):921-6. The binding of the inducer causes a change in the conformation of the two domains of the lac repressor. This will cause a change in the positions of the DNA-binding domains at the top, so that the afﬁnity for the lac operator is greatly reduced. Fig. 6-19 29 A low resolution structure of the tetrameric lac repressor bound to 2 operator sequences. The 4-helix bundle at the bottom ties the 4 subunits together. It is a ﬂexible joint. Crystal structure of the lactose operon repressor and its complexes with DNA and inducer. Lewis M, Chang G, Horton NC, Kercher MA, Pace HC, Schumacher MA, Brennan RG, Lu P. Science. 1996 Mar 1;271(5253):1247-54. 30 Transcriptional control of the lac operon is a simple example of signal integration (lactose and glucose), and combinatorial control (CAP combines with any different factors in various combinations). Fig. 18-6 When lactose is absent, repressor forms tetramers, and is an effective repressor of the lac operon. When lactose is present, in the absence of glucose, allolactose forms, binds to the repressor, its conformation changes, and it cannot bind to the operator. When glucose is present, cAMP levels are low, and since CAP cannot bind to the DNA unless cAMP is bound, CAP does not activate. Fig. 18-6 When glucose is absent, cAMP will be high, CAP will be bound to the DNA, and transcription is activated. Note by Francois Jacob Box 18-2 Fig. 3 Nobel Prize 1965: Monod Jacob Lwoff 1910-1976 1920-2013 1902-1994 Studies of the lac operon were used to develop ideas about control of gene expression 33 ▯ Transcriptional Regulation in Bacteria (II):▯ More Examples Friday, November 6, 2015 BIOL 5304 Lecture 27 Lessons from study of the lac operon: Allostery Lactose (allolactose) and cyclic AMP are effector molecules that bind to proteins and cause conformational changes that affect function. These are examples of allostery. How does lactose get into the cell, and how is it converted to allolactose, before induction occurs? The lac operon is always expressed at a low rate. The control is said to be “leaky”. Therefore, the permease that allows transport across the cellular membrane (lacY) and the β-galactosidase (lacZ) that converts lactose to allolactose are always present at some low level, even before induction. The binding of allolactose to lac repressor is an allosteric effect, since its binding does not compete directly with DNA binding, even though its effect is to decrease, or prevent, the binding of lac repressor to DNA. Similarly, cyclic AMP is an effector, and it also activates the expression of the lac operon. The level of cAMP is a consequence of the energy state of the bacterial cell. When glucose is plentiful, the level of cAMP decreases. The binding of cAMP to CAP is necessary for its binding to DNA. This binding allows CAP to enhance the binding of the RNA polymerase, which binds alongside it. The cAMP binding site in CAP is distinct from its DNA binding site, and so this is allostery. More lessons from the lac operon: signal integration Expression of the lac operon is controlled from two independent signals: lactose and cyclic AMP Cyclic AMP is also an example of combinatorial control. CAP works not only with the lac operon, but also with more than 100 other genes in E. coli. Typically, CAP will be working with other regulators, as it does in the lactose system. Eukaryotes tend to use signal integration and combinatorial control in even more complex ways. How did Monod, Jacob and their co-workers ﬁgure out how expression of the lac genes was controlled? They did not have the many tools of molecular biology that are available today. Their investigations started from the observation that when E. coli was grown in the presence of lactose, an enzyme, β-galactosidase appears. This enzyme allows E. coli to utilize the sugar lactose. Was this enzyme always present, and only needed to be activated? or did a new gene need to be expressed, and if so, how could that be accomplished? They were able to isolate mutants in which β-galactosidase was always expressed, regardless of the lactose level. The two classes of mutants were distinguished by introducing a segment of wild type DNA, containing the lac genes, into mutant cells. One class of mutants behaved as wild type under this situation. This class had defective repressors, and so the introduced genes could provide active repressors that were able to repress all of the lac ZYA genes. Now β-galactosidase is made only in the presence of lactose. Box 18-2 Fig. 1 i.e., the lacI gene acts in trans The other mutants were found in what came to be known as the operator. The change in DNA sequence did not allow the repressor to bind, but the RNA polymerase binding was not affected. Box 18-2 Fig. 2a The operator mutants functioned only in cis. The presence of a second, wild type operator, did not lead to normal control of expression of lacZYA. The mutant chromosome still did not bind repressor. These experiments lead to the idea that a factor must bind to the DNA near the operon. Box 18-2 Fig. 2b Tryptophan Operon The tryptophan operon is regulated differently because the genes need to be expressed only when tryptophan levels are low. Two different mechanisms are used in E. coli: 1. A conventional repressor 2. Attenuation Attenuation is a form of regulation ﬁrst described by CharlesYanofsky. It is described in Ch. 20, pp. 707-708. Abbott-ASM LifetimeAchievement 1998 National Medal of Science winner 2005 Charles Yanofsky (1925- ) TheAbbott-ASM LifetimeAchievementAward honors a distinguished scientist for a lifetime of outstanding contributions in fundamental research in any of the microbiological sciences. The 1998Award is presented to Charles Yanofsky, Ph.D., Professor, Department of Biological Sciences, Stanford University, in recognition of his pioneering discoveries that have become the cornerstones of molecular microbiology. His contributions span almost 50 years and are remarkable for their impact as well as the diverse areas of microbiology they affected. The award is sponsored byAbbott Laboratories. Yanofsky's career began with a M.S. and Ph.D. degrees in microbiology from Yale University. His ﬁrst major contribution was the identiﬁcation of several of the intermediates in the pathway of niacin synthesis from tryptophan in Neurospora crassa . Yanofsky's work continued to have an impact on the ﬁeld of microbial physiology with his elucidation of the previously unknown steps in the pathway of tryptophan biosynthesis in Neurospora and E. coli. This work provided critical insights into microbial metabolism upon which thousands of studies of different microorganisms now rely. In the 1960s, Yanofsky began work on the structure of proteins and genes responsible for tryptophan biosynthesis. He used the expression and regulation of these proteins as tools to dissect the inner workings of the cell's genetic apparatus. This work on the nature of mutations that prevent tryptophan biosynthesis produced some of Yanofsky's most signiﬁcant discoveries.According to Richard Losick, "No introductory course on molecular biology is complete without a presentation of Dr. Yanofsky's famous experiment proving the colinearity of the gene and its protein product. His remarkable exploitation of the E. coli tryptophan operon to work out the biochemical pathway of the synthesis of an amino acid and to elucidate fundamental aspects of transcription and translation, such as missense suppression and polarity, are historic achievements. His landmark discovery of the elegant mechanism of transcriptional attenuation is one of the most original contributions to the ﬁeld of gene control since the discovery of repression by Jacob and Monod." .... Yanofsky is a Member of the NationalAcademy of Sciences and a Fellow of theAmerican Academy of Microbiology. 10 ON THE COLINEARITY OF GENE STRUCTURE AND PROTEIN STRUCTURE. YANOFSKY C, CARLTON BC, GUEST JR, HELINSKI DR, HENNING U. Proc Natl Acad Sci U S A. 1964 Feb;51:266-72. No abstract available. One of the genes in the Trp operon, gene A, encodes a protein that is one subunit of an enzyme in the Tryptophan biosynthesis pathway. They took 16 mutants with alterations in gene A and mapped their locations by frequency of recombination. Then they isolated all of the proteins and mapped the locations of the alterations by protein sequencing. They established the colinearity of genes and proteins in this way. Altogether there are ﬁve genes necessary for the biosynthesis of tryptophan in the trp operon. When tryptophan levels are high, it binds to the repressor, and that complex binds to the operator and turns off expression of the trp genes. The leader region is part of the attenuation system. Box 20-1 Fig. 1 The leader encodes a short peptide with two Trp codons. This reading frame overlaps with segment 1. The transcriptional terminator is formed by segments 3 and 4. Box 20-1 Fig. 2 13 If Trp is available, translation of the leader peptide proceeds, and transcription terminates following the leader sequence, but prior to the trpE-A genes . Regions 3 and 4 form a stem and loop, and the polyU will cause termination of transcription as a rho-independent or intrinsic terminator. Box 20-1 Fig. 3a If Trp levels are low, translation of the leader peptide stalls at the two Trp codons, and this prevents segment 1 from base pairing with segment 2. Therefore, transcription does not terminate, because segments 2 and 3 will now base pair. Box 20-1 Fig. 3b i.e.,if translation stalls, transcription continues i.e.,if no ribosomes are present, transcription terminates 15 Other amino acid operons are regulated in a similar way. His and Leu are regulated by attenuation only, there is no repressor. Note that they have many codons in their leader sequences. Note: this type of attenuation can occur only if transcription and translation operate simultaneously. (i.e. not in eukaryotes) The tryptophan repressor is a homodimer. The 2 subunits are colored cyan and magenta, except for the helix-turn-helix motifs. The tryptophans are red. They contact the DNA. The HTH motifs are in the major groove, as usual, but in this case they do not make direct contacts with the bases, but water molecules mediate the interactions. This was a controversial ﬁnding. Tryptophan is a co-repressor, not an inducer. Crystal structure of trp repressor/operator complex at atomic resolution. Otwinowski Z, Schevitz RW, Zhang RG, Lawson CL, Joachimiak A, Marmorstein RQ, Luisi BF, Sigler PB. Nature. 1988 Sep 22;335(6188):321-9. Erra17m in: Nature 1988 Oct 27;335(6193):837. When tryptophan is plentiful, there are two ways in which transcription is repressed: When the tryptophan concentration is high enough to form complexes with the repressor, transcription of the biosynthesis genes will be blocked. If, however, some RNA polymerase is able to begin transcription, that will be terminated before the trp genes are transcribed, if tryptophan concentration is high. So, the attenuation mechanism is independent of the the trp repressor, and provides a second level of control over expression. Analysis of transcriptional control in bacteria by small effector molecules Outcomes: No transcription Basal level of transcription Elevated level of transcription Possible actions of small effectors: Binds to a repressor to disable it (inducer, e.g. allolactose)) Binds to a repressor to enable it (co-repressor, e.g., tryptophan) Binds to an activator to enable it (co-activator, e.g., cAMP) Binds to an activator to disable it (example not given) Effector molecules are not repressors or activators, instead they bind to those molecules. Activator by-pass experiments can demonstrate that binding is sufﬁcient for stimulating transcription. In part (a), different binding partners, X andY , achieve the same result as CAP and α. In part (b), CAP is fused to the RNA polymerase, and transcription is still stimulated. Box 18-1 Fig. 1 Other examples of regulation in bacteria Remember that E. coli primarily uses the sigma-70 factor, but that several other sigma factors exist. Other sigma-factors are used for the expression of subsets of genes, especially those that need to be expressed simultaneously. The sigma-32 factor is induced by heat shock, and it directs the transcription of a group of genes involved in coping with this stress. As its concentration increases in the cell it displaces the sigma-70 from the RNA polymerases, and this leads to expression of the heat shock genes. Alternative sigma factors can be used in sequence to direct a group of genes in an orderly fashion. host σ phage σ phage σ Phage SPO1, which infects Bacillus subtilis, directs the expression of its genes, by ﬁrst using the host sigma factor to transcribe genes it needs “early”, and then its own sigma factors to direct middle and late genes. Fig. 18-15 Not all activators work by simple recruitment. Some must induce conformational changes in other proteins, or in the promoter DNA. This is an allosteric mechanism: the binding of one factor induces a conformational change in another. The ﬁrst such activator we will look at is NtrC, which controls expression of genes involve in nitrogen metabolism. NtrC has an inactive/active transition similar to the lac repressor and CAP . Rather than the binding of an inducer, NtrC is activated by the action of an enzyme NtrB, a kinase. So, NtrC is activated by phosphorylation, which induces a conformational change. The inactive state is dimeric, (upper left), and the active state is hexameric, (upper right). The blue domains bind DNA (called the bottom). After ATP hydrolysis, the “top” side binds the sigma-54. The ATPase activity of the hexameric NtrC changes its conformation, and affects its interaction with the sigma-54-RNA polymerase. This allows the RNA polymerase to initiate transcription. The structural basis for regulated assembly and De Carlo S, Chen B, Hoover TR, Kondrashkina E,. Nogales E, Nixon BT. Genes Dev. 2006 Jun 1;20(11):1485-95. The NtrC binding sites to DNA are usually at some distance from the promoter, and have been shown to function as far as 1-2 kb away. This requires a large loop of DNA to form. NtrC is shown as a dimer, but upon phosphorylation by the kinase NtrB, it forms a hexamer as shown before. After ATP hydrolysis by NtrC, the RNA polymerase can begin transcription. Fig. 18-16 Some genes controlled by NtrC have a nearby site for a DNA bending protein called IHF (integration host factor, because of its role in the life of phage λ. Bending of the DNA can bring two regions into close proximity, such as an RNA Polymerase and an activator. Fig. 12-11 Summary of action of NtrC at the right. sigma 54 binds the RNA Pol to a promoter, but keeps it inactive. IHF bends the DNA to bring the NtrC closer to the RNA Pol, and to activate transcription. At the left, a sigma 70 at a different promoter, immediately proceeds to DNA opening. Structure, function, and tethering of DNA-binding domains in σ(54) transcriptional activators. Vidangos N, Maris AE, Young A, Hong E, Pelton JG, Biopolymers. 2013 Dec;99(12):1082-96 A second example of an activator the works by allosteric control rather than simple recruitment: MerR In this example, the protein MerR can change the conformation of the DNA in the promoter region for MerT. Without this conformational change the MerT promoter is nonfunctional. MerR must be activated by binding Hg ++ for activation to occur. The merT promoter elements are separated by 19 bp rather than the normal 15-17. This puts their faces on opposites sides of the duplex DNA. The merR activator (when Hg ++is bound) binds and distorts the DNA, such that the two faces of the promoter are 70 aligned, and can bind the RNA polymerase + σ factor. The dimeric MerR inserts an α-helix (magenta) into the major groove and bends the DNA. Notice the tilting of the bases. The structural mechanism for transcription activation by MerR family member multidrug transporter activation, N terminus. Newberry KJ, Brennan RG. J Biol Chem. 2004 May 7;279(19):20356-62 30 The effect of MerR on DNA structure is similar to eliminating 2 base pairs. Fig. 18-18 Not all repressors work by blocking the RNA polymerase. In E. coli the Gal repressor controls genes necessary for the utilization of the sugar galactose. It represses by binding near the Gal promoter. It interacts with the RNA polymerase and prevents it from entering the open complex. Therefore, the genes are not expressed. Another example is the P pr4tein from a bacteriophage (φ29) that binds near promoters and in general, activates transcription by also binding to the RNA polymerase. However, with a very strong promoter, the extra binding it provides to the RNA polymerase can prevent it from escaping. So, it can also act as a repressor. ▯ Transcriptional Regulation in Bacteria (III):▯ Bacteriophage λ Monday, November 9, 2015 BIOL 5304 Lecture 28 But ﬁrst a look at the control of arabinose utilization genes in bacteria Arabinose is another sugar, like lactose, which is a potential carbon source for bacteria. The presence of arabinose turns on expression of the araBAD genes, which allow the bacterium to metabolize arabinose. Expression of the araBAD genes is controlled by the activators CAP and araC. (a)These genes are expressed in the presence of arabinose and the absence of glucose. Fig. 18-19 When araC (golden) binds arabinose (black), it forms one kind of dimer that is able to bind to 2 adjacent sites, I and 1 I2, next to the RNA polymerase, which is activated. (a) Fig. 18-19 4 The araC protein has 2 domains, an arabinose binding domain and a DNA binding domain. It has 2 possible conformations, depending upon whether arabinose is bound or not. The arabinose ﬁts into one end of the barrel, and the magenta loop closes over it. (b) In the absence of arabinose, araC forms a different kind of dimer, and binds to site I and araO . This leaves the I site 1 2 2 vacant, and the loop prevents RNA Pol binding, so there is no activation of transcription. Fig. 18-19 6 Allosteric regulation by change of dimerization, as exemplified by the AraC protein from Escherichia coli. arabinose The structural basis of allosteric regulation in proteins. Laskowski RA, Gerick F, Thornton JM. 7 The N-terminal arms determine the way in which the monomers interact. So, AraC can be either an activator (+ arabinose), or a repressor. Functional modes of the regulatory arm of AraC. Rodgers ME, Holder ND, Dirla S, Schleif R. Proteins. 2009 Jan;74(1):81-91. 8 Introduction to E. coli Bacteriophage λ The life cycle of λ is well characterized, and many regulatory steps are understood. It has a linear chromosome of about 48,000 bp of DNA, and it can circularize through its self-cohesive ends (12 nucleotide overhangs). Once inside the bacterium it can propagate itself in 2 ways: 1. Replication by the host system, eventually resulting in lysis of the host, and release of ~100 phage particles ready to infect other cells. This is referred to as the “lytic mode”. 2. Integration into the host chromosome, where it can stay in- deﬁnitely. In this mode the phage DNA is replicated with the host chromosome. This is called the “lysogenic mode”. λ is now a “prophage” and the host is a “lysogen”. Events such as UV damage can trigger the phage to convert to the lytic mode. Two Pathways Fig. 18-20 Prophage Lytic growth is induced by damage or rapid growth. The λ genome has about 50 genes. Lysogenic growth requires the expression of only a few of them. Lytic growth requires the expression of most of the Fig. 18-21 genes. We will focus on an important regulatory region ﬁrst, which contains 3 promoters: PL L-leftward P RM-repressor maintenance RM PR R-rightward Fig. 18-21,22 There are 2 genes in this region: cro is transcribed from the P Rromoter during lytic growth. cI is transcribed from the P promoter under lysogenic growth. RM The P Lromoter is also active during lytic growth. Both P Lnd P aRe strong promoters and the RNA polymerase does not require any activators. PRM is a weak promoter and requires an activator. Fig. 18-23 How is this choice made? Cro (repressor) λ repressor This is a complex story of repressors and activators. Fig. 18-23 The cI gene encodes the λ repressor, a 2 domain protein shown schematically below. It can form oligomers. It must dimerize to bind DNA. It can function as an activator or as a repressor. It has a helix-turn-helix. Fig. 18-24 The N-terminal 92 amino acids of the λ repressor has been crystallized with a DNA segment. It formed a dimer in the crystal, as it would have naturally, had its C-terminal domain been present. The 2 subunits are colored cyan and greentint, with the Helix-Turn-Helix motifs in blue. They ﬁt into the major groove, as we have seen before. Refined 1.8 A crystal structure of the Beamer LJ, Pabo CO.erator complex. J Mol Biol. 1992 Sep 5;227(1):177-96. Structural tutorial 16 Cro (control of repressor and other things) functions only as a repressor. It has only one domain, and it binds to operator DNA as a dimer. Only one Cro subunit and half of an operator sequence are shown below. The HTH motif is shown in blue. It also binds in the major groove. Refined structure of Cro repressor protein from bacteriophage lambda suggests both flexibility and plasticity. Ohlendorf DH, Tronrud J Mol Biol. 1998 Jul 3;280(1):129-36. 17 Both Cro and λ repressor can bind to the same 6 operators. O R1 O R2 OR3 O L1 O L2 OL3 O R1 O R2 OR3 are associated with the rightward PromRter P and OL1 OL2 O L3are associated with the leftward PromLter, P Each of the operators is similar in sequence, about 17 bp, and each can bind a dimer of Cro or λ repressor. Because the sequences are slightly different, the afﬁnities are also different: Afﬁnity for λ repressorO > O ≈ O In each case R1 R2 R3 about 10-fold different. Afﬁnity for Cro: OR3> O R2 ≈ O R1 Arrangement of the 3 operator sites with respect to the rightward promoter P and the P promoter: R RM Fig. 18-25 The promoters are offset differently with respect to R2 Occupancy of λ repressor at O R1 blocks RNA polymerase at P aRd occupancy at O R3 blocks it at RM Occupancy at O R2blocks RNA polymerase at P R but recruits RNA polymerase at P RM Since the λ repressor binds O R1with highest afﬁnity, and the other 2 operators with equal but lower afﬁnity, how can O be ﬁlled, but not O ? R2 R3 Fig. 18-25 Cooperativity allows these 2 sites to ﬁlled, while the third remains empty. Remember that repressor can form tetramers. This arrangement allows both operator sites to be ﬁlled simultaneously, even though they have different intrinsic afﬁnities. The second dimer has 2 interaction surfaces now. The same opportunity is not available to the third site. Box 18-4 Fig. 1 Cooperativity allows the fraction of operator sites bound by repressor to be increased for any concentration of repressor. Fig. 18-26 Cro (product of cro) and λ repressor (product of cI) compete for the same operator sites. Fig. 18-25 22 Fig. 18-27 When λ repressor is expressed at a moderate level, it binds to both O R1 and O R2 as a tetramer. This represses the transcription of cro (to the right), and activates the transcription of cI (to the left). (positive autoregulation) This increases the level of λ repressor, until eventually the O site is also ﬁlled, and then R3 transcription of cI is also repressed (negative autoregulation). 23 When the level of Cro increases, and the level of λ repressor decreases, Cro will occupy the O site. This blocks R3 expression of cI (λ repressor) from P RM and allows transcription of cro from the strong promoter P . As the R concentration of Cro increases, eventually sites O and O R2 R1 will be occupied by Cro, and further expression will be blocked. Fig. 18-27 24 If lysogeny is established (λ repressor rules), how does induction (change to the lytic mode) occur? 25 RecA is involved in the response of E. coli cells to damage, in addition to its role in recombination. It has a proteolytic activity that can be activated. Its primary target is the LexA repressor, that normally represses numerous SOS genes, including DNA repair enzymes. It proteolyzes the LexA so that it cannot bind its operator. A similar proteolysis occurs with λ repressor, preventing it from forming oligomers, from binding its operators. RecA protease activity λ repressor LexA SOS genes ↳ The sudden loss of λ repressor means that P RM is no longer activated, and so expression of λ repressor stops. Transcription at the strong promoter P cRn proceed, and so Cro eventually builds up and lytic growth occurs. 27 Fig. 18-25 In recent years it was learned that this process is even more efﬁcient than previously thought, due to unexpected cooperativity between the Left and Right operators. This means that as the concentration of repressor drops, the occupancy of operators will drop even faster than it would without cooperativity. Fig. 18-28 Fig. 18-29 How monomers, dimers and tetramers interact to form the octamer. Top, 2 dimers have interacting surfaces, red (R) and blue(B). Lower left, interaction of 2 dimers. Lower right, interaction of 2 tetramer29 (No more surfaces available) The decision to enter lytic growth comes from this point. How is the initial choice made, when λ enters the cell? 30 Fig. 18-28 To understand that choice, we need to consider 2 additional genes: cII and cIII λ cIII is transcribed cII, like cro, is transcribed from promoter P from promoter P L R 31 Fig. 18-30 Fig. 18-31 cII is an activator for the RE promoter (repressor establishment) P is a weak promoter and needs activation. RE It transcribes the cI gene, but not cro or cII (the product of cI is λ repressor. It can be transcribed from 2 different promoters: P for establishment and P for maintenance.) RE RM 32 Since cro is transcribed from P , tRe initial direction is towards lysis. As cII is made, it can eventually tip the balance towards lysogeny. Fig. 18-31 33 If many phage particles infect a cell, it increases the chance of lysogeny. The more phage particles, the greater chance that one will establish lysogeny, and that will suppress the lytic pathway. This also makes sense for the phage, since if the multiplicity of infection is high, it indicates that the number of cells available to infect is low. Therefore, it may not be advantageous to lyse, and try to ﬁnd new cells to infect. to be continued … ▯ Transcriptional Regulation in Eukaryotes (III): Monday, November 16, 2015 BIOL 5304 Lecture 31 Combinatorial control is prevalent in eukaryotes. It makes complexity possible in gene regulation. Fig. 19-20 Factor 3 is used in combinations with other factors. The previous two examples of signal integration (in activation of the HO gene and β-interferon) are also examples of combinatorial control. Those activators are involved in the activation of other genes using Combinatorial control in yeast ensures that: a cells express a speciﬁc genes and α cells express α speciﬁc genes a cells must make the regulatory protein a1 α cells must make regulatory proteins α1 and α2 Both cell types must make regulatory protein Mcm1 Remember that yeast can grow as haploid cells of type a or type α, or as a diploid a/α. The interaction of the 4 regulatory proteins will generate 3 different expression patterns. The cell type is determined by the gene present at the MAT locus. Fig. 19-21 Combinatorial control in a cells a cells contain the a gene at the MAT locus, which codes for the a1 regulatory protein. Mcm1 is also expressed in a cells. a speciﬁc genes are activated by Mcm1. α speciﬁc genes are not activated. Haploid speciﬁc genes are expressed. Combinatorial control in α cells α cells contain the α genes at the MAT locus, which code for both the α1 and α2 regulatory proteins. Mcm1 is also expressed in α cells. a speciﬁc genes are repressed by dimeric α2 and Mcm1. α speciﬁc genes are activated by α1 and Mcm1. Haploid speciﬁc Combinatorial control in a/α diploid cells In diploid cells both a and α genes are at the MAT loci, but only a1 and α2 regulatory proteins are made. Mcm1 is also expressed in diploid cells. a speciﬁc genes are repressed by dimeric α2 and Mcm1. α speciﬁc genes are not activated. Haploid speciﬁc genes are repressed a1 and α2. The 4 regulatory proteins work together to generate 3 different expression patterns. Fig. 17-18 Analysis of different species of yeast reveals an evolutionary pathway of regulation: S. cerevisiae is brewer’s yeast C. albicans is a human pathogen They diverged 300-900 million years ago, about when ﬁsh and mammals diverged. But, they each have two mating types and share some aspects of regulation. Both use the heterodimer of a1/α 2 to repress haploid speciﬁc genes Fig. 17-18 Analysis of different species of yeast reveals an evolutionary pathway of regulation: In K. lactis, a1/α2 functions as a repressor of Rme1 Starvation activated Rme1, which then activates haploid speciﬁc genes, allowing the yeast to make a and α spores. Fig. 17-18 How do repressors work in eukaryotes? The typical bacterial repressor binds near the promoter and blocks the RNA polymerase from binding. Eukaryotes do not use this method, but use a variety of others. a. Competition with activators b. Inhibition of activators c. Direct interaction with the Transcription machinery d. Indirect repression by recruitment of other factors Fig. 19-22a The repressor can compete with an activator for the same site, or for an overlapping site. Other variations: The repressor can be the same as the activator, but without the activation domain. Or, for activators that must form dimers to bind, the repressor can bind directly to the activator, but lack a DNA-binding domain. In these cases, the rate of transcription will depend upon the concentrations of the proteins, and their afﬁnities. The repressor can bind near an activator and inhibit it, usually by blocking its activation domain. The activator can still bind, but it cannot activate. Fig. 19-22b The repressor can bind at a regulatory site and interact directly with the transcription machinery, for example, as shown here, the Mediator, and cause inhibition. Fig. 19-22c Fig. 19-23 Example: Repression of the GAL1 gene in yeast In between the promoter and the UAS, a protein called Mig1 binds, in the presence of glucose. Transcription of GAL1 is repressed when Mig1 recruits a complex that includes Tup1. Tup1 recruits a histone deacetylase, and/or interacts directly with the transcription machinery. Other experiments have shown that Tup1 can repress when bound to DNA upstream of genes, using different DNA binding proteins (i.e., like a bypass experiment). Fig. 19-22d The most common mechanism is indirect repression. The repressor binds at a nucleosome site and recruits a nucleosome modiﬁer of a type opposite to that of activators. For example, a chromatin compacter, or a histone deacetylase. Here the histone deacetylase is shown removing acetyl groups, leading to more compact nucleosomes, and less transcriptional activity. Signal Transduction and Transcriptional Regulation In bacteria, some small molecules act directly in transcriptional control as inducers or co-repressors. In eukaryotes, many molecules act indirectly with the transcriptional machinery through signal transduction pathways. We saw one example of this in bacteria. The NtrC activator is itself activated by phosphorylation in response to ammonia. A Typical Signal Transduction Pathway The signaling molecule typically binds to a membrane-bound cell surface receptor, outside of the cell. This triggers an event inside the cell, acting through the receptor. For example, it might be the activation of an enzyme. The signal is then transmitted through G-proteins, or kinases, to a transcriptional activator. The “activated” transcriptional activator then travels to the nucleus to ﬁnd the appropriate genes. Two signal transduction pathways from mammalian cells are shown on the next slide: STAT and Ras. STAT pathway Fig. 19-24 Ras pathway Phosphorylation of a transcription factor allows it to enter the nucleus and bind DNA. STAT pathway Fig. 19-24 The signaling molecule is called a cytokine. A kinase (JAK) is bound to the receptor inside the cell. Binding of the cytokine triggers the activation of the kinase, and it phosphorylates both the receptor and itself. The transcription factor STAT binds to the phosphorylated receptor, and becomes phosphorylated itself. This triggers Pthe release of phospho-STAT, which now can dimerize, enter to enter the nucleus and bind DNA. the nucleus and bind DNA to activate transcription. The signaling molecuSTAT pathway a tyrosine kFig. 19-24ptor, which dimerizes, self-phosphorylates, and recruits Grb2 and SOS. SOS activates Ras by acting as a nucleotide exchange factor. Ras releases GDP , then binds GTP . Activated Ras initiates the activation of the MAP kinase cascade (mitogen activated protein kinase). Ultimately, a transcription factor is phosphorylated, and enters the nucleus. Ras pathway How do Transcriptional Regulators in eukaryotes work? 1. Unmasking an Activating Region In bacteria, the activators and repressors are commonly regulated by changing their abilities to bind to DNA. This is not typically done in eukaryotes. One method is illustrated in the next slide: An activator, pre-bound to DNA, is unmasked. The masking protein is released, due to binding of a factor or ligand, or as a consequence of phosphorylation. The Gal4 activator is bound at the UAS, but it is prevented from activating because Gal80 is bound. Gal80 can bind galactose, and if so, it is released from Gal4, which is free to activate transcription. Fig. 19-25 Similar: Gal4 mammalian activator E2F is masked by Rb (retinoblastoma protein). Rb also recruits a histone deacetylase to further repress transcription. Phosphorylation of Rb releases it from E2F, allowing E2F to activate. How do Transcriptional Regulators in eukaryotes work? 2. Another method involves transport into or out of the nucleus. Only after the signaling pathway has functioned is the regulator allowed to leave the cytoplasm and enter the nucleus (e.g., STAT and Ras) It might be held in the cytoplasm by an inhibitor protein, or at the cellular membrane. Or it might be locked in a conformation that prevents it from being transported to the nucleus. Some Activators and Repressors come in Pieces The glucocorticoid receptor (GR, slide 6, lecture 30) is held in a complex with Heat shock protein 90 (Hsp90) until its ligand arrives (L). It binds to GRE sites, which come in 2 types. Some also bind the co-activator, CREB binding protein (CBP), and activate transcription. Others bind HDACs, and transcription is repressed. Cytoplasm
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