B M B 400 PART FOUR - V = Chapter 19. Regulation of eukaryotic genesB M B 400Part Four: Gene RegulationSection V = Chapter 19REGULATION OF EUKARYOTIC GENESA. Promoters1. Eukaryotic genes differ in their state of expressiona. Recall from Part One of the course that most genes in eukaryotes are notexpressed in any given tissue.Of the approximately 30,000 genes in humans, any particular tissue willexpress a few at high abundance (these are frequently tissue specific, e.g.globin genes in red cells) and up to a few thousand at low abundance (thesefrequently encode functions needed in all cells, i.e. "housekeeping genes."You can measure this by the kinetics of hybridization between mRNA andcDNA.b. The genes that are not expressed are frequently in an "inactive" region of thechromatin. The basic model is that genes that will not be expressed are keptin a default "off" state because they are packaged into a conformation ofchromatin that prevents expression.c. Expression of a gene then requires opening of a chromatin domain, followedby the steps discussed in Part Three of this course: assembly of atranscription complex. transcription, splicing and other processing events,translation, and any requisite post-translational modifications.d. Various active genes can be transcribed at distinctive rates, primarilydetermined by the differences in rate of initiation. This ultimately producesthe characteristic abundance of each mRNA, ranging from very high to verylow.2. Those genes that are expressed can be transcribed at a basal rate from the"basal” or “minimal” promoter, and in many cases they also can be induced toa high level of expression.The process of going from no expression to basal expression may differfundamentally from the process of going from basal expression to activatedhigh-level expression. For instance, for some genes the former could requirethat the strong negative effect of silencing chromatin be removed, whereas thelatter could involve covalent modification of particular transcriptional activators.However, the full mechanistic details of both processes are not yet known,although it is clear that several enzymatic activities, many of them composed ofmultiple polypeptide subunits, are involved in each. Changes in chromatinstructure and roles for transcriptional activators have been proposed in bothprocesses, so in fact there may be more similarity than one would have supposedinitially. The fact is that we simply do not know at this time. Addingcomplexity to ambiguity, one should realize that the mechanisms may differamong the many genes in an organism. Both processes (going from no expression to basal expression, and goingfrom basal to activated expression) are part of transcriptional activation,B M B 400 PART FOUR - V = Chapter 19. Regulation of eukaryotic geneswhich is currently an area of intense investigation in molecular genetics. Thus,even though a full understanding of this process eludes us, it is important toexplore what is currently known about gene regulation in eukaryotes, as well assome of the still-unanswered questions. That is what we will do in Chapters 19and 20.Figure 4.5.1. Expression states of promoters for RNA polymerase II. Each of these stateshas been described for particular genes, but it is not clear that all states are in one obligatorypathway. For instance, it possible that some gene activation events could go from silentchromatin to basal transcription without passing through open but repressed and pausedtranscription.B M B 400 PART FOUR - V = Chapter 19. Regulation of eukaryotic genesa. Basal transcription(1) Is frequently studied by in vitro transcription, using defined templatesand either extracts from nuclei or purified components.(2) Requires RNA polymerase with general transcription factors (e.g.TFIID, TFIIA, TFIIB, TFIIE, TFIIF, and TFIIH for RNA polymeraseII), as previously covered in Part Three.b. Activated transcription(1) Occurs via transcriptional activators interacting directly or indirectly withthe general transcription complex to increase the efficiency of initiation.(2) The transcriptional activators may bind to specific DNA sequences in theupstream promoter elements, or they may bind to enhancers (see SectionB below).(3) The basic idea is to increase the local concentration of the generaltranscription factors so the initiation complex can be assembled morereadily. The fact that the activators are bound to DNA that is close to thetarget (or becomes close because of looping of the DNA) means that thelocal concentration of that protein is high, and hence it can boost thelocal concentration of the interacting general transcription factors.3. Stalled polymerasesa. RNA polymerase will transcribe about 20 to 40 nucleotides at the start ofsome genes and then stall at a pause site. The classic example are heat-shock genes in Drosophila, but other cases are also known.b. These genes are activated by release of stalled polymerases to elongate. Inthe case of the heat shock genes, this requires heat shock transcription factor(HSTF). The mechanism is still under study; some interesting ideas are(1) Phosphorylation of the CTD of the large subunit of RNA polymerase IIcauses release to elongation ("promoter clearance"). One candidate (butnot the only one) for the CTD kinase is TFIIH.(2) Addition of a processivity factor (analogous to E. coli Nus A?), maybeTFIIS.B M B 400 PART FOUR - V = Chapter 19. Regulation of eukaryotic genesB. SilencersSilencers are cis-acting regulatory sequences that reduce the expression from a promoter ina manner independent of position or orientation - i.e. they have the opposite effect of anenhancer. Two examples are the silencers that prevent expression of the a or α genes at thesilent loci of the mating type switching system in yeast and silencers at telomeres in yeast.The silencers work by sequence specific proteins, such as Rap1, binding to DNA inchromatin. These proteins serve as anchors for expansion of repressed chromatin. They dothis by recruiting silencing proteins called SIR proteins, named for their activity as silentinformation regulators. The SIR proteins assemble the chromatin into a large complex thatis not transcribed. In this complex, the H3 and H4 histones in the nucleosomes havehypoacetylated N-terminal tails, the DNA can be methylated, and the entire silencedcomplex is resistant to DNase digestion in vitro, all of which are characteristic ofcondensed, closed chromatin. The large