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Chapter 17

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  1. Chapter 17 Gene Regulation in Eukaryotes

  2. In eukaryotic cells, expression of a gene can be regulated at all those steps we saw in bacteria, and a few additional ones besides. • In many cases, a given transcript can be spliced in alternative ways to generate different products, and this too can be regulated. • The eukaryotic transcriptional machinery is more elaborate than its bacterial counterpart.

  3. Nucleosomes and their modifier influence access to genes. • Many eukaryotic genes have more regulatory binding sites and are controlled by more regulatory proteins than are typical bacterial genes. • Promoter • Regulatory binding sites • Regulatory sequences: the stretch of DNA encompassing the complete collection of regulator binding sites for a given gene.

  4. In multicellular organisms , regulatory sequences can spread thousands of nucleotides from the promoter-both upstream and downstream –and can be made up of tens of regulator binding sites. Often these binding sites are grouped in unites called enhancers, and a given enhancer binds regulators responsible for activating the gene at a given time and place. Alternative enhancers bind different groups of regulators and control expression of the same gene at different times and places in response to different signals. (Figure 17-1)

  5. FIGURE 17-1The regulatory elements of a bacterial, yeast, and human gene.

  6. OUTLINE • Conserved Mechanisms of Transcriptional Regulation from Yeast to Mammals • Recruitment of Protein Complexes to Genes by Eukaryotic Activators • Signal Integration and Combinatorial Control • Transcriptional Repressors

  7. Signal Transduction and the Control of Transcriptional Regulators • Gene “Silencing” by Modification of Histones and DNA • Eukaryotic Gene Regulation at Steps after Transcription Initiation • RNAs in Gene Regulation

  8. CONSERVED MECHANISMS OF TRANSCRIPTIONAL REGULATION FROM YEAST TO MAMMALS

  9. Many of the basic features of gene regulation are the same in all eukaryotes. • This is tested using a reporter gene. The reporter gene consists of binding sites for the yeast activator inserted upstream of the promoter of a gene whose expression level is readily measured.

  10. The typical eukaryotic activator works in a manner similar to the simplest bacterial case. • In contrast , repressors work in a variety of ways ,some different from anything we encountered in bacteria. • Gene silencing, in which modification to regions of chromatin keep genes in sometimes large stretches of DNA switched off.

  11. Activators Have Separate DNA Binding and Activation Functions • Eukaryotic activators have separate DNA binding and activating regions as well. The two surfaces are very often in separate domains of the protein. (Figure 17-2)

  12. FIGURE 17-2 Gal4 bound to its site on DNA.

  13. One such gene is called GAL1. GAL4 binds to four sites located 275bp upstream of GAL1(Figure 17-3). • The separate DNA binding and activating regions of Gal4 were revealed in two complementary experiments.

  14. FIGURE 17-3 The regulatory sequences of the yeast GAL1 gene.

  15. FIGURE17-4 Domain swap experiment.

  16. Eukaryotic Regulators Use a Range of DNA-Binding Domains, but DNA Recognition Involves the Same Principles as Found in Bacteria

  17. One class of eukaryotic regulatory protein presents the recognition helix as part of a structure very like the helic0turn-helix domain; others present the recognition helix within quite different domain structures. • In eukaryotes bind DNA as heterodimers, and in some cases even as monomers.

  18. Homeodomain proteins. The homeodomain is a class of helix-turn-helix DNA-binding domain and recognizes DNA in essentially the same way as those bacterial proteins (Figure 17-5).

  19. FIGURE 17-5 DNA recognition by a homeodomain.

  20. FIGURE 17-6 Zinc finger domain. • Zinc containing DNA-Binding Domains. Zinc finger proteins, Zinc cluster domain.

  21. FIGURE 17-7 Leucine zipper bound to DNA. • Leucine zipper motif. This motif combines dimerization and DNA-binding surfaces within a single structural unit.

  22. Helix-loop-helix proteins. An extended αhelical region from each of two monomers insets into the major groove of the DNA. Leucine zipper and HLH proteins are often called basic zipper and basic HLH proteins : this is because the region of the αhelix that binds DNA contains basic amino acid residues (Figure 17-8).

  23. FIGURE 17-8 Helix-loop-helix motif.

  24. Activating Regions Are Not Well-Defined Structures • It is believed that activating regions consist of reiterated small units, each of which has a weak activating capacity on its own. Each unit is a short sequence of amino acids, the greater the number of units, and the more acidic each unit, the stronger the resulting activating region.

  25. RECRUITMENT OF PROTEIN COMPLEXES TO GENES BY EUKARYOTIC ACTIVATORS

  26. Activators Recruit the Transcriptional Machinery to the Gene

  27. Eukaryotic activators rarely, if ever, through a direct interaction between the activator and RNA polymerase. First, the activator can interact with parts of the transcription machinery other than polymerase, and, by recruiting them, recruit polymerase as well. Second, activators can recruit nucleosome modifiers that alter chromatin in the vicinity of a gene and thereby help polymerase bind.

  28. According to one view, most of the machinery comes to the gene in a single , very large complex called the holoenzyme, which contains the mediator , RNA polymerase , and some of the general transcription factors (Figure 17-9).

  29. FIGURE 17-9 Activation of transcription initiation in eukaryotes by recruitment of the transcription machinery.

  30. Recruitment can be visualized using the technique called chromatin immunoprecipitation (ChIP ). • In activator bypass experiments, activation is observed when RNA polymerase is recruited to the promoter without using the natural activator –polymerase interaction (Figure 17-10).

  31. FIGURE 17-10 Activation of transcription through direct tethering of mediator to DNA.

  32. Activators also Recruit Nucleosome Modifiers that Help the Transcription Machinery Bind at the Promoter

  33. In activator bypass experiments, activation is observed when RNA polymerase is recruited to the promoter without using the natural activator –polymerase interaction (Figure 17-10).

  34. FIGURE 17-11 Local alterations in chromatin structure directed by activators.

  35. Action at a Distance: Loops and Insulators • Various models have been proposed to explain how proteins binding in between enhancers and promoters might help activation in the cells of higher eukaryotes. In Drosophila, a protein called Chip aids communication between enhancer and gene.

  36. In eukaryotes, chromatin may in some places form special structures that actively bring enhancers and promoters closer together. • If an enhancer activates a specific gene 50kb away , what stops it from activating other gene whose promoters are within that range? Specific elements called insulator control the actions of activators (Figure 17-12).

  37. FIGURE 17-12 Insulators block activation by enhancers.

  38. In other assays, insulators also seem able to inhibit the spread of chromatin modifications. • Silencing is a specialized form of repression that can spread along chromatin, switching off multiple genes without the need for each to bear binding sites for specific repressors. Insulator elements can block this spreading, so insulators protect genes from both indiscriminate activation and repression.

  39. Appropriate Regulation of Some Groups of Genes Requires Locus Control Regions

  40. There are five different globin genes in humans (Figure 17-13a). Although clustered, these genes are not all expressed at the same time. Rather, the different genes are expressed at different stages of development. • A group of regulatory elements collectively called the locus control region, or LCR, is found 30-50 kb upstream of the whole cluster of globin genes (Figure 17-13).

  41. FIGURE 17-13 Regulation by LCRs.

  42. SIGNAL INTEGRATION AND COMBINATORIAL CONTROL

  43. Activators Work Together Synergistically to Integrate Signals

  44. When multiple activators work together, they do so synergistically. Two activators can recruit a single complex. • Cooperativity: Synergy can also result from activators helping each other bind under conditions where the binding of one depends on binding of the other (Figure 17-14). • Synergy is critical for signal integration by activators.

  45. FIGURE 17-14 Cooperative binding of activators.

  46. Signal Integration: the HO Gene Is Controlled by Two Regulators; One Recruits Nucleosome Modifiers and the Other Recruits Mediator • The yeast S. cerebisiae divides by budding. We will focus here on the expression of a gene called HO. The HO gene is expressed only in mother cells and only at a certain point in the cell cycle.

  47. FIGURE 17-15 Control of the HO gene.

  48. Signal Integration: Cooperative Binding of Activators at the Human β-Interferon Gene • The human β-interferon gene is activated in cells upon viral infection. Infection triggers three activators: NFkB, IRF, Jun/ATF. The structure formed by these regulators bound to the enhancer is called an enhanceosome (Figure 17-16).

  49. FIGURE 17-16 The human β-interferon enhanceosome.

  50. The binding of the activators is cooperative for two reasons. First, the activators interact with each other. second, an additional protein, called HMG-I, binds within the enhancer and aids biding of the activators by bending the DNA in a way that facilitates the interactions among them.