Control of Gene Expression in Bacteria Gene Regulation

Control of Gene Expression in Bacteria Gene Regulation and Information Flow: an overview Metabolizing Lactose—A Model System: multiple genes, classes of mutants; the lac operon model and the discovery of the repressor (Jacob and Monod) Catabolite Repression and Positive Control: How Does Glucose Influence Formation of the CAP-c. AMP Complex? The Operator and the Repressor—an Introduction to DNABinding Proteins: Finding the Operator; DNA Binding via the Helix-Turn-Helix Motif How Does the Inducer Change the Repressor’s Affinity for DNA?

17. 1 Gene Regulation and Information Flow • Escherichia coli has served as an excellent model organism for the study of prokaryotic gene regulation because, like most bacteria, it can use a wide array of carbohydrates to supply carbon and energy. • Producing all the enzymes required to process all the various carbohydrates all the time would waste energy. It is logical to predict that the enzymes E. coli produces match the sugars that are available at a given time. • Efficient use of resources, via tight control over gene expression, is critical for E. coli's survival.

• Glucose is the preferred carbon source for E. coli. Lactose is used only when glucose is depleted. • E. coli produces high levels of b-galactosidase, the enzyme that cleaves lactose to glucose + galactose, only when lactose is present in the environment. Thus, lactose acts as an inducer—a molecule that stimulates the expression of a specific gene. • Jacques Monod found that b-galactosidase is not expressed in E. coli cells grown in medium containing glucose or glucose + lactose but only in medium containing lactose and no glucose

Master plates containing medium with many sugars were replica-plated to medium with lactose as the only sugar to screen for colonies that could not grow on lactose

• Indicator plates allow mutants with metabolic deficiencies to be observed directly. Colonies grown on lactose were sprayed with ONPG (o-nitrophenol-b. D-galactoside), an indicator with a structure similar to that of lactose. When bgalactosidase breaks down ONPG, the intensely yellow compound onitrophenol is produced, turning colonies bright yellow.

Indicator plates allow mutants with metabolic deficiencies to be observed directly. Colonies grown on lactose were sprayed with ONPG (o-nitrophenol-β-D-galactoside), an indicator with a structure similar to that of lactose. When β-galactosidase breaks down ONPG, the intensely yellow compound o-nitrophenol is produced, turning colonies bright yellow. Three classes of mutants were found:

The lac Operon • Jacob and Monod coined the term operon for a set of coordinately regulated bacterial genes that are transcribed together into one m. RNA, usually encoding proteins that work together. • The group of genes involved in lactose metabolism was termed the lac operon. • The lac operon genes are expressed on a single polycistronic m. RNA, and their expression is regulated by a single promoter. • The repressor does not physically block RNA polymerase from contacting the promoter but instead prevents transcription initiation by keeping RNA polymerase from unwinding the DNA helix.

The Impact of the lac Operon Model • The lac operon model introduced the idea that gene expression is regulated by physical contact between regulatory proteins and regulatory sites within the DNA. • Negative control occurs when something must be taken away for transcription to occur. • The lac operon repressor exerts negative control over three protein-coding genes by binding to the operator site in DNA near the promoter. For transcription to occur, an inducer molecule (a derivative of lactose) must bind to the repressor, causing it to release from the operator

Superimposed positive control: Inhibition of a metabolic pathway by its breakdown products (end-product inhibition) is called catabolite repression (e. g. , glucose inhibiting the lactose operon).

• When little glucose is available and less ATP is produced, a derivative of ATP called cyclic AMP (c. AMP) is produced. • c. AMP allows activation of expression of the lac operon through binding to the catabolite activator protein (CAP), which binds a DNA sequence called the CAP binding site located just upstream of the lac promoter. • Binding by the positive regulator CAP strengthens the lac promoter to increase expression.

Finding the operator site in the DNA: DNA footprinting is used to identify DNA sequences that are bound by regulatory proteins.

• The section of a helix-turn-helix regulatory protein that binds inside the DNA major groove is called the recognition sequence; it recognizes and interacts with the specific nitrogenous bases there.

The active lac repressor is a tetramer of four Lac. I+ monomers, each of which can bind to an operator sequence to block the opening of the double helix for transcription. One tetramer can bind two operator sequences and cause the DNA between the operators to “kink” or “loop” When the repressor interacts with the inducer (either lactose or IPTG, an analog of lactose), the inducer binds to a central region of the repressor and induces a change in the shape of the repressor tetramer, making it release the DNA.

Vibrio cholerae – a bacterial pathogen

Regulation of toxin gene expression in cholera: pg. 378 -9 Note: this group of genes is not in most Vibrio cholera bacteria; it is brought in on a particular temperate phage that becomes a prophage. A second temperate phage, this one of the filamentous family, is responsible for the pilus that binds the bacteria in place in the small intestine as its toxin stimulates human adenyl cyclase, leading to massive secretion of chloride ions and thus water, producing the potentially-lethal severe diarrhea.

Eukaryotic Regulation: • There are 4 primary differences between gene expression in bacteria and eukaryotes: • (1) Packaging of DNA (chromatin structure) in eukaryotes; • (2) splicing of m. RNA in eukaryotes; • (3) complexity of transcriptional control in eukaryotes; • (4) coordinated expression through operons in bacteria.

Alternate m. RNA splicing in different tissue types: • At least 35% of human genes undergo alternative splicing. Although we have only around 40, 000 genes, it is anticipated that we express between 100, 000 and 1 million different protein products.

Experiment carried out in reticulocytes (young red blood cells) Ovalbumin gene β-globin gene (hemoglobin subunit)

• Histone acetyl transferases (HATs) add negatively charged acetyls (acetylation) or methyls (methylation) to histones. This decondenses the chromatin and allows gene expression. Histone deacetylases (HDACs) remove the acetyl groups from histones to allow chromatin condensation and turn off gene expression.

p 53 – Guardian of the Genome and Tumor Suppressor

The signal transducers and activators of transcription (STATs) are an excellent example of post- translational regulation via protein phosphorylation

The gene encoding the splicing factor SF 2/ASF is a proto-oncogene Rotem Karni, Elisa de Stanchina, Scott W Lowe, Rahul Sinha, David Mu & Adrian R Krainer krainer@cshl. edu Nature Structural & Molecular Biology - 14, 185 - 193 (2007) • • • Alternative splicing modulates the expression of many oncogene and tumor-suppressor isoforms. We have tested whether some alternative splicing factors are involved in cancer. We found that the splicing factor SF 2/ASF is upregulated in various human tumors, in part due to amplification of its gene, SFRS 1. Moreover, slight overexpression of SF 2/ASF is sufficient to transform immortal rodent fibroblasts, which form sarcomas in nude mice. We further show that SF 2/ASF controls alternative splicing of the tumor suppressor BIN 1 and the kinases MNK 2 and S 6 K 1. The resulting BIN 1 isoforms lack tumor-suppressor activity; an isoform of MNK 2 promotes MAP kinase–independent e. IF 4 E phosphorylation; and an unusual oncogenic isoform of S 6 K 1 recapitulates the transforming activity of SF 2/ASF. Knockdown of either SF 2/ASF or isoform-2 of S 6 K 1 is sufficient to reverse transformation caused by the overexpression of SF 2/ASF in vitro and in vivo. Thus, SF 2/ASF can act as an oncoprotein and is a potential target for cancer therapy.