Connecting Concepts Across Chapters 9

The focus of this chapter has been on how the flow of information from genotype to phenotype is controlled. We have seen that there are a number of potential points of control in this pathway of information flow, including changes in gene structure, transcription, mRNA processing, mRNA stability, translation, and posttranslational modifications.

Gene regulation is critically important from a number of perspectives. It is essential to the survival of cells, which cannot afford to simultaneously transcribe and translate all of their genes. The evolution of complex genomes consisting of thousands of genes would not have been possible without some mechanism to selectively control gene expression. Gene regulation is also important from a practical point of view. A number of human diseases are caused by the breakdown of gene regulation, which produces proteins at inappropriate times or places. Gene regulation is also important to genetic engineering, where the key to success is often not getting genes into a cell, which is relatively easy, but getting them expressed at useful levels. For all of these reasons, there is tremendous interest in how gene expression is controlled, and understanding gene regulation is one of the frontiers of genetic research.

Information presented in this chapter builds on the foundation of molecular genetics developed in Chapters 10 through 15. The mechanisms of gene regulation provide important links to several topics in subsequent chapters. Gene regulation is important to the success of recombinant DNA, which is discussed in Chapter 18. Gene regulation also plays an important role in the genetics of development and cancer, which are discussed in Chapter 21.

CONCEPTS SUMMARY]_

• Gene expression may be controlled at different levels, including the alteration of gene structure, transcription, mRNA processing, RNA stability, translation, and posttranslational modification. Much of gene regulation is through the action of regulatory proteins binding to specific sequences in DNA.

• Genes in bacterial cells are typically clustered into operons— groups of functionally related structural genes and the sequences that control their transcription. Structural genes in an operon are transcribed together as a single mRNA.

• In negative control, a repressor protein binds to DNA and inhibits transcription. In positive control, an activator protein binds to DNA and stimulates transcription. In inducible operons, transcription is normally off and must be turned on; in repressible operons, transcription is normally on and must be turned off.

• The lac operon of E. coli is a negative inducible operon that controls the metabolism of lactose. In the absence of lactose, a repressor binds to the operator and prevents transcription of the structural genes that encode p-galactosidase, permease, and transacetylase. When lactose is present, some of it is converted into allolactose, which binds to the repressor and makes it inactive, allowing the structural genes to be transcribed and lactose to be metabolized. When all the lactose has been metabolized, the repressor once again binds to the operator and blocks transcription.

• Positive control in the lac operon and other operons is through catabolite repression. When complexed with cAMP, the catabolite activator protein (CAP) binds to a site in or near the promoter and stimulates the transcription of the structural genes. Levels of cAMP are indirectly correlated with glucose; so low levels of glucose stimulate transcription and high levels inhibit transcription.

• The trp operon of E. coli is a negative repressible operon that controls the biosynthesis of tryptophan.

• Attenuation is another level of control that allows transcription to be stopped before RNA polymerase has reached the structural genes. It takes place through the close coupling of transcription and translation and depends on the secondary structure of the 5' UTR sequence.

• Small RNA molecules, called antisense RNA, are complementary to sequences in mRNA and may inhibit translation by binding to these sequences, thereby preventing the attachment or progress of the ribosome.

• Transcriptional control regulates the lytic and lysogenic cycles of bacteriophage X. The transcription of certain operons stimulates the transcription of some operons and represses the transcription of others. Which operons are stimulated and which are repressed depends on the affinity of promoters for repressor and activator proteins.

• Like gene regulation in bacterial cells, much of eukaryotic regulation is accomplished through the binding of regulatory proteins to DNA. However, there are no operons in eukaryotic cells, and gene regulation is characterized by a greater diversity of mechanisms acting at different levels.

• In eukaryotic cells, chromatin structure represses gene expression. During transcription, chromatin structure may be altered by the acetylation of histone proteins and demethylation.

• The initiation of eukaryotic transcription is controlled by general transcription factors that assemble into the basal transcription apparatus and by transcriptional activator proteins that stimulate normal levels of transcription by binding to regulatory promoters and enhancers.

• Some DNA sequences limit the action of enhancers by blocking their action in a position-dependent manner.

• Coordinately controlled genes in eukaryotic cells respond to the same factors because they have common response elements that are stimulated by the same transcriptional activator.

• Gene expression in eukaryotic cells may be influenced by RNA processing.

• Gene expression may be regulated by changes in RNA stability. The 5' cap, the coding sequence, the 3' UTR, and the poly(A) tail are important in controlling the stability of eukaryotic mRNAs. Proteins binding to the 5' end of eukaryotic mRNA may affect its translation.

• RNA silencing takes place when double-stranded RNA is cleaved and processed to produce small interfering RNAs that bind to complementary mRNAs and bring about their cleavage and degradation.

• Control of the posttranslational modification of proteins also may play a role in gene expression.

[important terms gene regulation (p. 000) induction (p. 000) structural gene (p. 000) regulatory gene (p. 000) regulatory element (p. 000)

domain (p. 000) operon (p. 000) regulator gene (p. 000) regulator protein (p. 000) operator (p. 000)

negative control (p. 000) positive control (p. 000) inducible operon (p. 000) inducer (p. 000) allosteric protein (p. 000)

repressible operon (p. 000) corepressor (p. 000) coordinate induction (p. 000) partial diploid (p. 000) constitutive mutation (p. 000)

catabolite repression (p. 000) catabolite activator protein

(CAP) (p. 000) adenosine-3', 5'-cyclic monophosphate (cAMP) (p. 000) attenuation (p. 000)

attenuator (p. 000) antiterminator (p. 000) antisense RNA (p. 000) transcriptional antiterminator protein (p. 000) DNase I hypersensitive site (p. 000)

chromatin-remodeling complex (p. 000) CpG island (p. 000) coactivator (p. 000) insulator (p. 000) heat-shock protein (p. 000) response element (p. 000)

SR protein (p. 000) RNA silencing (p. 000) small interfering RNAs (siRNAs) (p. 000)

Worked Problems

1. A regulator gene produces a repressor in an inducible operon. A geneticist isolates several constitutive mutations affecting this operon. Where might these constitutive mutations occur? How would the mutations cause the operon to be constitutive?

An inducible operon is normally not being transcribed, meaning that the repressor is active and binds to the operator, inhibiting transcription. Transcription takes place when the inducer binds to the repressor, making it unable to bind to the operator.

Genotype of strain

(d) lacI+ lacP+ lacO+ lacZ— lacY— / lacI— lacP+ lacO+ lacZ+ lacY+

Constitutive mutations cause transcription to take place at all times, whether the inducer is present or not. Constitutive mutations might occur in the regulator gene, altering the repressor so that it is never able to bind to the operator. Alternatively, constitutive mutations might occur in the operator, altering the binding site for the repressor so that the repressor is unable to bind under any conditions.

; 2. For E. coli strains with the lac genotypes, use a plus sign ( + ) to ; indicate the synthesis of p-galactosidase and permease and a minus sign ( — ) to indicate no synthesis of the enzymes.

Lactose absent Lactose present

P-Galactosidase Permease P-Galactosidase Permease

Solution

Genotype of strain

(d) lacI+ lacP+ lacO+ lacZ— lacY—/ lacI— lacP+ lacO+ lacZ+ lacY+

Lactose absent

Lactose present

P-Galactosidase Permease P-Galactosidase Permease

(a) All the genes possess normal sequences, and so the lac operon functions normally: when lactose is absent, the regulator protein binds to the operator and inhibits the transcription of the structural genes, and so p-galactosidase and permease are not produced. When lactose is present, some of it is converted into allolactose, which binds to the repressor and makes it inactive; the repressor does not bind to the operator, and so the structural genes are transcribed, and p-galactosidase and permease are produced.

(b) The structural lacZ gene is mutated; so p-galactosidase will not be produced under any conditions. The lacO gene has a constitutive mutation, which means that the repressor is unable to bind to it, and so transcription takes place at all times. Therefore, permease will be produced in both the presence and the absence of lactose.

(c) In this strain, the promoter is mutated, and so RNA polymerase is unable to bind and transcription does not take place. Therefore p-galactosidase and permease are not produced under any conditions.

(d) This strain is a partial diploid, which consists of two copies of the lac operon—one on the bacterial chromosome and another on a plasmid. The lac operon represented in the upper part of the genotype has mutations in both the lacZ and lacY genes, and so it is not capable of encoding p-galactosidase or permease under any conditions. The lac operon in the lower part of the genotype has a defective regulator gene, but the normal regulator gene in the upper operon produces a diffusible repressor (trans acting) that binds to the lower operon in the absence of lactose and inhibits transcription. Therefore no p-galactosidase or permease is produced when lactose is absent. In the presence of lactose, the repressor cannot bind to the operator, and so the lower operon is transcribed and p-galactosidase and permease are produced.

3. The fox operon, which has sequences A, B, C, and D, encodes enzymes 1 and 2. Mutations in sequences A, B, C, and D have the following effects, where a plus sign ( + ) = enzyme synthesized and a minus sign (—) = enzyme not synthesized.

Fox absent

Fox present

Enzyme 1

Enzyme 2

Enzyme 1

Enzyme 2

Mutation in sequence

No mutation A B C D

(a) Is the fox operon inducible or repressible?

(b) Indicate which sequence (A, B, C, or D) is part of the following components of the operon:

Regulator gene Promoter

Structural gene for enzyme 1 Structural gene for enzyme 2

(a) When no mutations are present, enzymes 1 and 2 are produced in the presence of Fox but not in its absence, indicating that the operon is inducible and Fox is the inducer.

(b) Mutation A allows the production of enzyme 2 in the presence of Fox, but enzyme 1 is not produced in the presence or absence of Fox, and so A must have a mutation in the structural gene for enzyme 1. With B, neither enzyme is produced under any conditions, and so this mutation most likely occurs in the promoter and prevents RNA polymerase from binding. Mutation C affects only enzyme 2, which is not produced in the presence or absence of lactose; enzyme 1 is produced normally (only in the presence of Fox), and so mutation C most likely occurs in the structural gene for enzyme 2. Mutation D is constitutive, allowing the production of enzymes 1 and 2 whether or not Fox is present. This mutation most likely occurs in the regulator gene, producing a defective repressor that is unable to bind to the operator under any conditions.

Regulator gene Promoter

Structural gene for enzyme 1 Structural gene for enzyme 2

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