15 Regulation of Gene Expression

1. 15 Regulation of Gene Expression Expression of genetic information is dependent on regulatory mechanisms that either activate or repress the transcription of genes. Transcription is modulated by theinteraction at various regulatory molecules with DNA sequences, most often located upstream from affected genes.Genetic regulation in eukaryotes also occurs during post transcriptional events.

2. Translation • Initiation • Elongation • Termination Gene expression • Transcription • Initiation Elongation Termination Francois Jacob Jacques Monod

3. 15.1Genetic Regulation in Prokaryotes:An Overview15.2Lactose Metabolism in E. coli: An Inducible SystemStructural GenesThe Discovery of Regulatory MutationsThe Operon Model: Negative'ControlGenetic Proof of the Qperon ModelIsolation of the lac RepresserThe CAP Protein: Positive Control of the lac Operon • 15.3Tryptophan Metabolism in E. coli:A Repressible Gene SystemGenetic Evidence for the trp Operon ,Attenuation15.4Genetic Regulation in Eukaryotes:An Overview

4. 15.5Regulatory Elements, Transcription Factors, and Eukaryotic GenesPromotersEnhancersTranscription FactorsStructural Motifs of Transcription FactorsAssembly of the Transcription ComplexChromatin Conformation, DNA Methylation,and Gene Expression15.6Gene Regulation by Steroid Hormones15.7Posttranscriptional Regulation of GeneExpression:Alternative Splicing of mRNA

5. 15.1Genetic Regulation in Prokaryotes:An Overview • Regulation of gene expression has been studied extensively in prokaryotes, particularly in Escherichia coll. Highly efficient mechanisms have evolved that turn genes on and off, depending on the cell's metabolic needs in particular environments. Detailed analysis of proteins in E. coll has shown that for the more than 4000 polypeptide chains encoded by the genome, a vast range of concentration of gene products exists. Some proteins may be present in as few as 5-10 molecules per cell, whereas others, such as ribosomal proteins and the many proteins involved in the glycolytic pathway, are present in as many as 100,000 copies per cell. The idea that microorganisms regulate the synthesis of gene products can be illustrated by considering the utilization of lactose (a galactose-glucose-containing disaccha-ride) as a carbon source. When it is present in the growth medium, many bacteria and yeast produce enzymes specific to lactose metabolism. When lactose is absent, the enzymes are not manufactured. These organisms thus "adapt" to their environment, producing certain enzymes only when specific chemical substrates are present. Such enzymes are said to be inducible, reflecting the role of the substrate, which serves as the inducer of enzyme production. In contrast, other enzymes that are produced continuously, re-gardless of the chemical makeup of the environment, are described as constitutive.Studies have also revealed cases where the presence of a specific molecule causes inhibition of genetic expression. This is often the case for molecules that are the end products of biosynthetic pathways. Amino acids can be synthesized by bacterial cells, but if the amino acids are present in the growth medium, they can be taken up and used. In such cases, it is inefficient for the cell to produce the enzymes necessary for the synthesis of those amino acids, and transcription of mRNA for the appropriate biosynthetic enzymes is repressed. This is an example of a repressible system of gene regulation.Regulation, whether it is inducible or repressible, may be under either negative or positive control. Under negative control, genetic expression occurs unless it is shut off by some form of a regulator molecule. In contrast, under positive control, transcription occurs only if a regulator molecule directly stimulates RNA production. In theory, either type of control can govern inducible or repressible systems. Our discussion in the ensuing sections of this chapter will clarify these contrasting systems of regulation. For the enzymes involved in lactose and tryptophan, negative control is operative.

6. Regulation of Gene Expression • At Transcription stage: • Economical, but can’t be reversed quickly • At Translation stage: • Wasteful, but easily reversible

7. Regulation of Gene Expression Feedback regulation: • Positive control: the gene is off unless it is turned on • Negative control: the gene is on unless it is turned off

8. Regulation of Gene Expression Feedback regulation: • Induction: the “target” molecule turns expression ON (e.g., by disactivating the repressor) • Repression: the “target” molecule turns expression OFF

9. The lac Operon in E. coli Genetic System under both Induction and Repression regulation. Let’s first consider Induction.

19. Cis- and trans- acting regulatory sequences • Operator: a cis-acting element Influences expression of genes downstream from it on the same 2-stranded DNA • Repressor gene: a trans- acting regulatory gene Influences expression of any relevant genes in the same cell, on the same or different DNA

20. Let’s stop here and solve some problems • You have 2 constitutive lac mutants (always expressing lac operon). You transform them with a plasmid containing an intact lac operon. One of them changes the phenotype to wild type. The other does not no matter how much you try. What may be the genotypes of these 2 mutants?

21. Let’s stop here and solve some problems • You have a mutant strain with a mutation in lac operon repressor gene, which prevents lactose from binding to repressor protein. • What will be the phenotype? Can it be changed by transforming this strain with a plasmid containing any lac operon structural or regulatory genes?

22. Homework! • Textbook, Ch. 17, p.459: #5, 6, 10(extra spicy, optional) • Problem manual, p.82-83: #44, 48 (solved)

23. Pop-up quiz:

27. CAP-cAMP control Glucose present -> cAMP level decreases -> no CAP-cAMP complex is formed -> no CAP binding to promoter -> no activation of transcription

29. Let’s compare two controls of lac operon • Repressor-operator control • CAP-cAMP control

30. Let’s compare two controls of lac operon • Repressor-operator control Negative control Control molecule: lactose • CAP-cAMP control Positive control Control molecule: glucose

31. Another operon under double control: Trp operon Trp = triptophan

35. Let’s compare regulation of of lac operon and trp operons • lac operon Induction (target molecule, lactose, induces expression by desactivating repressor protein) • trp operon Repression (target molecule, triptophan, represses expression by activating repressor protein)

36. But.. It’s not the whole story! Attenuation regulation of trp-operon

39. Regulation of Gene Expression in Prokaryotes Hartwell pp. 551-560, 567-571

40. Regulation of transcription • Differences in the basepairs in the -35 and -10 boxes allow genes to be expressed at different levels. • This kind of regulation is important, but it does not allow the cell to adjust its pattern of gene expression in a dynamic manner to meet changing needs and environmental stresses.

41. Strategiesfor regulating gene expression in prokaryotes • Switches in the s subunit of RNA polymerase • Control by a regulated repressor of transcription • Control by a regulated activator of transcription • Regulated attenuation (termination) of transcripts

42. Strategiesfor regulating gene expression in prokaryotes • Switches in the s subunit of RNA polymerase • Control by a regulated repressor of transcription • Control by a regulated activator of transcription • Regulated attenuation (termination) of transcripts

43. Switches in the s subunit of RNA polymerase • Under certain conditions, the cell needs to induce a large set of genes that are normally silent • Examples include: • heat-shock proteins • nitrogen starvation • developmental changes such as sporulation

44. s70 - s32 - s54- Switches in the s subunit of RNA polymerase • Under certain conditions, the cell needs to induce a large set of genes that are normally silent • This can be accomplished by synthesizing or activating a different RNA polymerase s subunit that recognizes a distinct set of promoter sequences. pg. 786

45. Strategiesfor regulating expression in prokaryotes • Switches in the s subunit of RNA polymerase • Control by a regulated repressor of transcription • Control by a regulated activator of transcription • Regulated attenuation (termination) of transcripts

46. In prokaryotes, genes with related functions can be expressed as a single mRNA controlled by one promoter Promoter Transcription P O LacZ LacY LacA lactose permease unknown function b-galactosidase The lac operon

47. Operons are regulated by two kinds of elements RNAP Transcriptional regulatory proteins Transcription Promoter Operator Gene 1 Regulatory sites

48. Transcriptional regulatory proteins • Often called transcription factors • Some transcriptional regulatory proteins stimulate transcription initiation (called activators) • Other transcriptional regulatory factors inhibit transcription (called repressors) • In bacteria, the sites where transcription factors bind reside close to the transcription start site. These binding sequences are called operators.

49. The regulatory gene for an operon can reside at another site of the chromosome pg. 869

50. The Lac Operon:A classic example of dynamic regulation of gene expression • b-galactosidase pg. 868