Express yourself! Gene expression

1. Express yourself!Gene expression

2. Key Concepts • In bacteria, gene expression can be controlled at three levels: transcription, translation, or post-translation (protein activation). • Changes in gene expression allow bacterial cells to respond to environmental changes. • Transcriptional control can be negative or positive. Negative control occurs when a regulatory protein prevents transcription. Positive control occurs when a regulatory protein increases the transcription rate.

3. Introduction • A cell does not express all of its genes all of the time. Instead, they are very selective about the genes they express, how strongly they are expressed, and when they are expressed. • Gene expression occurs when a gene product is actively being synthesized and used in a cell. Regulation of gene expression is critical to the efficient use of resources and thus survival.

4. Gene Regulation and Information Flow • Escherichia coli has served as an excellent model organism for the study of prokaryotic gene regulation. • Like most bacteria, E. coli can use a wide array of carbohydrates to supply carbon and energy. Control of gene expression allows E. coli to respond to its environment and switch its use of sugars. • Gene expression in bacteria was predicted to be triggered by specific signals from the environment.

5. Mechanisms of Regulation―An Overview • Information flow occurs in three steps, represented by arrows: DNA mRNA protein activated protein Remember what this was called??? • Genes can be under transcriptional, translational, or post-translationalcontrol. • All three types of regulation occur in bacteria.

6. Transcriptional Control of Gene Expression • Transcriptional control occurs when the cell does not produce mRNA for specific enzymes. • The cell avoids the production of these enzymes by utilizing regulatory proteins that prevent RNA polymerase from binding to a promoter. DNA mRNA protein activated protein x

7. Translational Control of Gene Expression • Translational control allows the cell to prevent the translation of an mRNA molecule that has already been transcribed. This can occur through many mechanisms: • Regulatory molecules can speed up mRNA degradation. • Translation initiation can be altered. • Translation proteins can be affected. DNA mRNA protein activated protein x

8. x Post-Translational Control of Gene Expression • Post-translational control occurs when the cell fails to activate a manufactured protein by chemical modification. • Chemical modification is an enzymatic process. Can be as simple, yet energy expensive as phosphorylation. Signal cascade during a signal transduction pathway is an example. DNA mRNA protein activated protein

9. Control of Gene Expression in Bacteria • All three forms of gene expression control occur in bacteria. • Transcriptional control is slow but efficient. • Translational control allows a cell to quickly change which proteins are produced. • Post-translational control provides the most rapid response but is energetically expensive.

11. Mechanisms of Regulation―An Overview • The level of expression of different genes can be highly variable. • Variation in gene expression allows cells to respond to changes in their environment.

12. Metabolizing Lactose―A Model System • E. coli’spreferred carbon source is glucose, and uses lactose only when glucose is depleted. • Before it can utilize lactose, E. coli must transport it into the cell, where the enzyme b-galactosidase can cleave it to produce glucose and galactose. • E. coli produces high levels of b-galactosidase only when lactose is present in the environment. • Thus, lactose acts as an inducer—a molecule that stimulates the expression of a specific gene.

13. Identifying Genes under Regulatory Control • To find the genes that code for -galactosidase and the membrane transport protein that brings lactose into the cell, Monod and François Jacob isolated and analyzed E. coli mutants that could not metabolize lactose.

14. Finding Mutants for a Particular Trait • Isolating mutants with respect to a particular trait is a two-step process. • Generate a large number of individuals with mutations at random locations in their genomes. • Use genetic screening on the mutants to find individuals with defects in the process or pathway in question.

15. Replica Plating to Find Mutant Genes • Replica plating, a process used to identify mutant cells, is a four-step process. • Grow bacterial colonies on master plates containing a medium with many sugars. • Transfer cells from each colony to a piece of sterilized velvet. • Transfer cells to a plate with a medium containing only lactose to screen for colonies that could not grow on lactose. This new plate is called a replica plate. • Compare the colonies on the master plate and replica plate.

16. Replica Plating to Find Mutant Genes • Indicator plates can also be used. • These allow researchers to directly observe mutants with metabolic deficiencies. • These plates made it possible for them to identify colonies without functioning β-galactosidase.

17. Different Classes of Lactose Metabolism Mutants • The three genes involved in lactose metabolism were named lacZ, lacY, and lacI. • Three classes of E. coli mutants defective in lactose metabolism were identified: • lacZmutants lack functional b-galactosidase. • lacY mutants lack the membrane protein galactoside permease and so cannot transport lactose into the cell. • lacI mutants are called constitutivemutants because they produce b-galactosidase and galactoside permease even when lactose is absent.

19. Several Genes Are Involved in Metabolizing Lactose • The lacZ and lacY genes code for proteins involved in lactose metabolism, while the lacI gene product serves a regulatory function. • In the absence of lactose, the lacI gene product shuts down expression of lacZ and lacY. When lactose is present, however, transcription of lacZ and lacY is induced. • Further studies revealed that the lacZ, lacY, and lacI genes are located close together on the circular E. coli chromosome. This suggested that lacI could control both lacZ and lacY.

21. Mechanisms of Negative Control: The Repressor • Transcription can be regulated via negative control or positive control. • Negative control occurs when a regulatory protein binds to DNA and shuts down transcription. • Positive control occurs when a regulatory protein binds to DNA and triggers transcription.

24. Mechanisms of Negative Control: The Repressor • Szilard and Monod hypothesized that the lacI gene codes for a repressor that exerts negative control over the lacZ and lacY genes. • They hypothesized that the repressor would bind directly to the DNA on or near the promoter for the lacZ and lacY genes. • Lactose then would interact with the repressor in a way that makes the repressor release from its binding site. • Lactose thus would induce transcription by removing negative control.

26. The lac Operon • Jacob and Monod coined the term operon for a set of coordinately regulated bacterial genes that are transcribed together into one mRNA. • The group of genes involved in lactose metabolism was thus termed the lac operon. • Later a fourth gene, called lacA, was discovered to be part of the lac operon. The lacA gene codes for the enzyme transacetylase, which has a protective function.

27. The lac Operon • Three hypotheses are central to the Jacob-Monod model of lac operon regulation: • The lacZ, lacY, and lacA genes are transcribed together and are thus coordinately regulated. • The lacI protein is a repressor that prevents transcription of the lac operon by binding to a site called the operator. • Lactose, the inducer, binds directly to the lacI repressor, causing it to release from the operator and ending negative control of the operon.

29. Why Has the lac Operon Model Been So Important? • Regulation of the lac operon provided an important model system in genetics. • We now know that gene expression of many bacterial operons is regulated by physical contact between regulatory proteins and specific regulatory sites on DNA. • In addition, as in the lac operon, the activity of many other key regulatory proteins is regulated by post-translational control.

30. Mechanisms of Positive Control: Catabolite Repression • Transcription of the lac operon is greatly reduced when glucose is present, even when lactose is also available. • When glucose is already available, the cell does not need to produce more by cleaving lactose. • This is an example of catabolite repression. • Occurs when one of the product molecules (the catabolite) of a reaction represses the production of the enzyme(s) responsible for that reaction. • In the case of the lac operon, glucose is the catabolite.

32. The CAP Protein and Binding Site • The absence of glucose activates expression of the lac operon through positive control. • The catabolite activator protein (CAP) binds the CAP binding site near the lac promoter and triggers transcription. • CAP binding strengthens the lac promoter to increase expression. • CAP is regulated by cyclic AMP (cAMP) binding to it. Only when CAP is bound to cAMP can it bind DNA. • If cAMP levels are low, CAP is not active and transcription is not increased.

34. Glucose Influences Formation of the CAP-cAMP Complex • When extracellular glucose concentrations are high, intracellular cAMP concentrations are low; when extracellular glucose concentrations are low, intracellular cAMP concentrations are high. • The enzyme adenylylcyclase produces cAMP from ATP and is inhibited by extracellular glucose. • The amount of cAMP and the rate of lac transcription are inversely related to the concentration of glucose.

36. Summary of Control of the lac Operon • Because positive and negative control elements are superimposed, E. coli fully activates the genes for lactose metabolism only when lactose is available and when glucose is scarce or absent.