STUDY HINTS
The problem of how genes are turned on and off at the proper time is a fascinating one. Geneticists still have a lot to learn in this area. Particularly in eukaryotic cells, the interaction between the nucleus and cytoplasm and the coordinated activation of functionally related genes at different times or in different tissues make development a complicated process, even in the simplest organisms. The operon model in prokaryotes is probably not directly analogous to the control systems of higher organisms, but it is an excellent place to begin getting the feel of the logic of regulatory systems.
There are two main types of operons: inducible and repressible. Inducible operons are normally turned off, since a repressor protein is bound at the operator site (O) and thus blocks RNA polymerase, which binds at the promoter site (P). Active transcription is blocked. Inducible operons can be activated by some substrate (the inducer) that binds with, and deforms, the repressor protein. Typically the inducer is some substance that is acted upon by the enzymes coded in the operon, so that the operon is turned on only when its products are needed by the cell. The repressor gene (i) may be quite distant from the operon, but the promoter and operator must be adjacent to the structural genes (SG1, SG2, etc.) that they control. The key is that the represser gene’s diffusible product is synthesized in an active form.
We can contrast this arrangement with that for a repressible operon. As with the inducible operon, function is controlled by an operator site that interacts with a repressor. However, unlike the inducible operon, the repressor protein is initially synthesized in an inactive form. It is activated by a sufficient buildup of the end product made by the enzymes that are coded for by the operon. The end product binds with the inactive repressor to produce an active repressor that turns off the operon. Thus a repressible operon is generally turned on until the product it codes for is in sufficient quantity. A key difference between the two types of operons is therefore the initial activity or inactivity of the repressor protein.
Mutations in the operator or in the repressor (sometimes called the regulator gene) can lead to constitutive activity – that is, to continuous transcription. For example, if the repressor protein is structurally abnormal, it will be unable to bind with the operator of an inducible operon, and the operon will be active even in the absence of the inducer. Alternatively, if the operator gene mutates to a form that will not allow binding of a repressor, as shown in the following figure, the operon will show constitutive activity.
Patterns of operon action can therefore be described largely in terms of the nature of the repressor protein and of the operator DNA sequence to which it binds. For example, mutations at the inducer binding site or mutations that interfere with the transition from an active to an inactive repressor protein shape will lead to an uninducible state whether the inducer is present or not. Mutations in the promoter gene or the structural genes can also lead to the absence of transcription and subsequent misexpression of one or more genes in the operon, even if other components are normal.
In eukaryotes, complex transformations in the physical state of the proteins that associate with DNA (i.e., chromatin) occur as a result of intracellular and extracellular signaling and are linked to chromosome replication, chromosome segregation during cell division, and gene expression. These changes in state involve reversible modifications to both DNA and the proteins that bind to DNA. The mechanisms that transduce alterations in gene expression in response to extracellular cues are just beginning to be understood at a molecular level. It is clear, however, that the general lessons learned from bacterial systems have relevance in eukaryotes. Vertebrate steroid hormones, for example, influence gene expression by attaching to a specific receptor. This interaction effectively produces a transcription factor that binds to a specific DNA sequence and promotes the recruitment of the proteins required for mRNA transcription. The use of genetics and biochemistry to dissect particular signaling pathways, has, as in bacteria, proved extremely valuable. The basic features of many signal transduction pathways are remarkably conserved in eukaryotes. How these pathways interact and are adapted to respond to environmental stimuli in particular developmental contexts is now the subject of intense study.
In prokaryotes, mRNA can be translated even while it is being produced. But the partitioning of transcription from translation in eukaryotes by the nuclear envelope allows a larger number of regulatory opportunities than in prokaryotes. Some of the levels at which the modification or manipulation of gene expression can occur in eukaryotes are: (1) transcriptional control, initiating gene expression to produce an RNA transcript; (2) processing control, which includes alternate intron splicing, addition of the 5′ cap and 3′ poly-A tail; (3) transportation control, regulating the flow of molecules like mature mRNA through the nuclear pores; (4) translational control, which can include competition for ribosome binding or the masking of mRNA in the cytoplasm; (5) posttranslational modification of the polypeptide after it is produced, such as binding with an inducer or having two or more polypeptides link together to form the functional protein; and (6) RNA stability, which affects the longevity of the message and thus the number of times it is used by the cell.
IMPORTANT TERMS
Allosteric proteins
Attenuation
Canalized characters
Catabolite repression
Constitutive mutation
Cytoplasmic determinant
Differentiation
Enhancer
Epigenesis
Epigenetic change
Genomic imprinting
Histones
Homeotic mutant
Homeobox
Hox genes
Imaginal discs
Operator
Operon
Polar mutation
Preformation
Promoter
Trans-acting factors
Transcription factor
PROBLEM SET 18
1. What is the major difference between an inducible operon and repressible operon in Escherichia coli?
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2. Various theories have been advanced to account for the extreme specialization exhibited by animal tissues. For example, it was suggested that all genes were lost from a cell during development except those genes expressed in each kind of cell. Another theory was that all of the original genes were present but that specialization involves imposing controls whereby particular genes are activated or repressed in different tissues. R. Briggs and T. J. King (1952) demonstrated the first strong evidence for one of these theories. Which theory was supported, and what was the evidence?
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3. The turnover rates of mRNA differ considerably when prokaryotes are compared with eukaryotes. In prokaryotes, many mRNAs function for only about 2–5 minutes, whereas in eukaryotes many function for several hours or more. It has been suggested that the turnover rate of specific mRNAs may be genetically determined by the gene that codes for the mRNA. What conclusions can you draw about the relationship between mRNA turnover rates and the adaptive responses that prokaryotes and eukaryotes show to their environments?
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4. In the African clawed toad, Xenopus, individuals homozygous for the anucleolate mutant die. These embryos produce what appear to be normal blastulas and gastrulas, and they form neural tissue, but they cannot go on to form normal tadpoles. They die early. How can you explain the delayed “time of action” or expression of the anucleolated gene?
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5. Assume that it is possible to produce a chromosomal aberration in E. coli in which the structural genes for one operon (histidine synthesis) replace the structural genes of the lactose operon, which normally codes inducibly for β-galactosidase permease, and gal acetylase. Normally, the histidine operon is repressible by an end product. The new operon would result in histidine synthesis that is
(a) induced by lactose,
(b) inhibited by excess histidine,
(c) inhibited by excess lactose,
(d) induced by excess histidine.
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6. Consider the following inducible operons:
Suppose you supply the inducer of operon 1 for only a short time. Also assume the inducer and repressor have a short half-life. Starting with the first operon turned on and the second operon turned off, how would you describe the continuing action of these two operons after the original (exogenous) inducer has been used up?
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7. Referring to the arrangement in problem 6, if the first operon is supplied with its inducer by some artificial means, what is the continuing action of these two operons?
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8. Again referring to the system in problem 6, if the inducer for operon 2 is supplied exogenously, what is the continuing action of the two operons?
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9. Protein hormones tend to work more rapidly than steroid hormones. One has its effect by inducing transcription, and the other acts by posttranslational modification. Which is which?
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10. Some steroid hormones bind to a cytoplasmic receptor that is then responsible for moving the complex into the nucleus. This part of the process is an example of what level of eukaryotic regulation?
(a) transcriptional control;
(b) processing control;
(c) transportation control.
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11. The proteolytic enzyme pepsin is secreted into the stomach in an inactive form called pepsinogen. There it is activated by HCl, and it cannot normally damage the cells that produced it. What kind of eukaryotic regulation does this example illustrate?
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12. Chromatin conformation differs between genes that are active and genes that are inactive. When genes are being actively transcribed, the structure of the chromatin associated with them changes, leaving the DNA bound to that chromatin more susceptible to degradation by DNAseI. Consider the following experiment. In mammals, the hormone insulin is produced by the beta cells in the pancreas and released into the bloodstream. Factor VIII, a protein required for blood clotting, is also released into the bloodstream, but it is synthesized by the liver. A rat is killed, and islet cells of the pancreas and hepatocytes from the liver are removed by dissection. Chromatin is then biochemically purified from both the pancreatic- and liver-cell nuclei. Each chromatin preparation is then treated with increasing concentrations of DNAseI (from 0 to 2 μg/ml). The DNA from the chromatin in these two tissues is then purified away from protein and subsequently cut with restriction enzymes. The restriction enzymes will cut the rat DNA so that a portion of the insulin gene will be found on a 2.5 kb restriction fragment, and the gene for factor VIII will be found on a 4.0 kb restriction fragment. Each DNA series is then run on electrophoretic gels, the DNA denatured and transferred to filter paper (Southern blot, Chapter 20), and hybridized with radioactively labeled single-stranded DNA from the insulin and factor VIII genes. The following two autoradiographs (A and B) are produced:
Which autoradiograph represents the sample prepared from pancreatic nuclei, and which represents the pattern prepared from liver nuclei? What is the basis for the difference in the patterns observed in these two Southern blots?
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13. Prader-Willi syndrome and Angelman syndrome are two human disorders that are associated with the inheritance of a small deletion on the long arm of chromosome 15. The characteristic symptoms of these disorders are quite different from one another and are easily distinguished clinically. Interestingly, when the deletion chromosome is inherited from the father, the child will have Prader-Willi syndrome. When the deletion chromosome is inherited from the mother, the child will have Angelman syndrome. Molecular analysis has shown that this region of chromosome 15 displays different DNA methylation patterns (differences in the addition of methyl groups to nucleotides, thereby preventing the expression of the gene). The pattern depends on whether chromosome 15 is inherited from the mother or the father. What might be happening to genes in this region of the genome that could account for the differences between the phenotypes associated with these two syndromes? (Hint: Consider phenomena listed under Important Terms.)
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ANSWERS TO PROBLEM SET 18
1. In inducible operons, transcription occurs only in the presence of a substrate. In repressible operons, on the other hand, transcription is initiated in the absence of the end product, and repression occurs when the end product has been built up to a sufficient level.
2. Briggs and King demonstrated the constancy of the genome (our second theory). Their experiment involved the successful transplantation of a somatic-cell nucleus from a blastula cell into an enucleated egg of the leopard frog Rana pipiens. They found that the recipient egg could develop into a complete, normal individual. Thus the somatic cells must have had a full complement of genes.
3. The rapid adaptation that prokaryotes, such as bacteria, show to changing environments depends on their ability to turn genes on and off in a rapid response. There would be little adaptive advantage to a rapid genetic response if the mRNA that it produced persisted for hours. In eukaryotes, however, much of the homeostatic response to changes in the environment is accomplished by cells and tissues showing specialized arrays of proteins. Under these conditions of epigenetic regulation, many mRNA species could be more efficient (or at least not maladaptive) if they functioned longer. Other factors might include protein half-life and rate and duration of mRNA synthesis.
4. The anucleolated genotype is deficient for some of the ribosomal RNA genes, and the loss of these rRNA molecules prevents the formation of functional ribosomes. During early stages of development, the embryos are using the large number of maternal ribosomes that are produced during oogenesis. New rRNA and new ribosomes normally appear at the gastrula stage, and this is where the lethality is expressed. Since the embryo cannot make new ribosomes, it loses its ability to make new proteins, and it dies.
5. The aberration described in this problem could be represented as shown in the figure on p. 163. The lactose operon (II) is inducible; that is, that it will be turned on by the presence of lactose in the cell. Even though the histidine operon is normally repressible by an end product, it is the activity of the operator that determines the action of the operon. The new operon has the inducible OII, and histidine synthesis would therefore be induced by lactose. The correct answer is (a).
6. If the first operon is turned on at the beginning of the problem, it will synthesize the first two enzymes and a repressor of operon 2. Since the inducer of operon 1 is gradually used up, operon 1 will eventually turn off. The repressor of operon 2 will no longer be produced, and operon 2 will be turned on. This will yield a new inducer for operon 1, and the two operons will cycle on and off.
7. If the first operon is supplied with its inducer constantly, the lack of inducer synthesis by the second operon is unimportant. Thus operon 1 will stay turned on. Operon 2 will not be turned on.
8. If the inducer for operon 2 were supplied exogenously, synthesis of enzymes 6 and 7 would begin, and inducer of operon 1 would be produced. This would turn on operon 1, which would then begin transcribing mRNA, leading to the synthesis of enzymes 1 and 2. In addition, repressor of operon 2 would be produced. If inducer for operon 2 were supplied exogenously, it should block the action of the repressor of 2. Both operons should then be on continuously.
9. Protein hormones act by posttranslational modification. The hormone binds to a membrane-bound receptor that activates a second messenger like cyclic-AMP inside the cell. This, in turn, activates proteins already present in an inactive form, posttranslational modification. This is a faster process than the numerous steps required by a typical steroid hormone, which binds to a receptor in the cytoplasm or nucleus to produce a transcription factor that then activates transcription. After processing, the mature mRNA is transported into the cytoplasm where it is finally translated to produce the gene product. Thus, steroid hormone effects take longer to complete.
10.
(c) Transportation control is the best answer, although the mechanism may involve an element of posttranslational modification.
11. This is an example of posttranslational modification, in which HCl alters the protein after its initial synthesis.
12. Autoradiograph A represents the liver tissue nuclei and autoradiograph B represents the pancreatic cell nuclei. When chromatin isolated from liver tissue (autoradiograph A) is treated with increasing concentrations of nuclease, the active gene (detected as a 4.5 kb band by the factor VIII gene probe) is degraded more rapidly than the inactive gene. The opposite situation is observed in autoradiograph B. In this tissue, it is the insulin gene (detected as a 2.5 kb band by the insulin gene probe) that is active and is more rapidly degraded. The factor VIII gene is inactive in pancreatic tissue, is in a different chromatin configuration, and is more resistant to nuclease digestion.
13. The region of chromosome 15 important in these two syndromes undergoes an epigenetic modification known as genomic imprinting. It appears that at least two different genes are involved in the Prader-Willi and Angelman syndromes, and they are differentially inactivated (perhaps through methylation of sequences important for transcriptional control) during normal gametogenesis. In males, certain portions of chromosome 15 are normally inactivated during spermatogenesis and passed on to the embryo in an inactive form. Similarly, different portions of chromosome 15 are normally inactivated during oogenesis in females. If the embryo now inherits a chromosome that is deletedfor the inactivated gene from the appropriate parent, no functional copies of the gene are present, resulting in either one syndrome or the other. For Prader-Willi syndrome, for example, the situation would be as follows:
The inactivating modifications that are transmitted to the fertilized egg are passed on to all somatic cells of an embryo. They are subsequently erased and reset in the developing germline, according to the sex of the embryo.