XII. Gene Regulation

- Overview: All cells in an organism contain the same genetic information; the key to tissue specialization is gene regulation – reading some genes in some cells and other genes in other cells.

Also, organisms can respond to their environment at a genetic level, so there must be a way for the environment to stimulate or repress the action of certain genes.

And changes occur through time, creating developmental changes And changes occur through time, creating developmental changes. We will look at how gene expression is regulated in these cases.

- Overview: Some Terminology: some enzymatic genes are only turned on if the substrate is present; this is an inducible system and the substrate is the inducer. Obviously, this is highly adaptive, as the cell saves energy by only producing the enzyme when it is needed.

- Overview: Some Terminology: some enzymatic genes are only turned on if the substrate is present; this is an inducible system and the substrate is the inducer. Obviously, this is highly adaptive, as the cell saves energy by only producing the enzyme when it is needed. some enzymes are on all the time, and are only turned off if a compound (often the product of the metabolic process they are involved with) is present. This is a repressible system, and the compound is the repressor. This is also adaptive, and the cell saves on enzymes if the product is already present.

- Overview: Some Terminology: some enzymatic genes are only turned on if the substrate is present; this is an inducible system and the substrate is the inducer. Obviously, this is highly adaptive, as the cell saves energy by only producing the enzyme when it is needed. some enzymes are on all the time, and are only turned off if a compound (often the product of the metabolic process they are involved with) is present. This is a repressible system, and the compound is the repressor. This is also adaptive, and the cell saves on enzymes if the product is already present. Constitutive genes are on all the time.

XII. Gene Regulation A. The lac Operon in E. coli

XII. Gene Regulation A. The lac Operon in E. coli When lactose is present, E. coli produce three enzymes involved in lactose metabolism. Lactose is broken into glucose and galactose, and galactose is modified into glucose, too. Glucose is then metabolized in aerobic respiration pathways to harvest energy (ATP). When lactose is absent, E. coli does not make these enzymes and saves energy and amino acids. How do they KNOW? : )

XII. Gene Regulation A. The lac Operon in E. coli As you remember, an “operon” was a region of genes that are regulated as a unit – it typically encodes > 1 protein involved in a particular metabolic pathway.

XII. Gene Regulation A. The lac Operon in E. coli As you remember, an “operon” was a region of genes that are regulated as a unit – it typically encodes > 1 protein involved in a particular metabolic pathway.

XII. Gene Regulation A. The lac Operon in E. coli Lac Y - permease – increases absorption of lactose

XII. Gene Regulation A. The lac Operon in E. coli Lac Y - permease – increases absorption of lactose Lac Z – B-galactosidase – cleaves lactose into glucose and galactose

XII. Gene Regulation A. The lac Operon in E. coli Lac Y - permease – increases absorption of lactose Lac Z – B-galactosidase – cleaves lactose into glucose and galactose Lac A – transacetylase – may code for enzymes that detoxify waste production of digestion.

XII. Gene Regulation The lac Operon in E. coli 1960 – Jacob and Monod proposed that this was an inducible system because the presence of the substrate INDUCES transcription. Repressor Gene Operator Repressor RNA Poly

XII. Gene Regulation The lac Operon in E. coli 1960 – Jacob and Monod proposed that this was an inducible system because the presence of the substrate INDUCES transcription. LACTOSE

The binding of lactose changes the shape of the repressor (allosteric reaction) and it can’t bind to the operator. XII. Gene Regulation The lac Operon in E. coli 1960 – Jacob and Monod proposed that this was an inducible system because the presence of the substrate INDUCES transcription. LACTOSE

XII. Gene Regulation The lac Operon in E. coli Mutant analyses confirmed these results:

XII. Gene Regulation The lac Operon in E. coli Mutant analyses confirmed these results:

XII. Gene Regulation The lac Operon in E. coli Mutant analyses confirmed these results:

XII. Gene Regulation The lac Operon in E. coli Mutant analyses confirmed these results: Curiously, there are only about 10 repressor molecules in each cell and they were not actually isolated and identified for 6 years (Gilbert).

XII. Gene Regulation The lac Operon in E. coli But it is even more complicated… if glucose AND lactose are present, the operon is OFF. This is adaptive, because it’s glucose the cell needs. If glucose is present, there is no need to break lactose down to get it. BUT HOW?

XII. Gene Regulation The lac Operon in E. coli But it is even more complicated… if glucose AND lactose are present, the operon is OFF. This is adaptive, because it’s glucose the cell needs. If glucose is present, there is no need to break lactose down to get it. BUT HOW? This involves a repressible pathway.

XII. Gene Regulation The lac Operon in E. coli Within the promoter, there is a binding site for Catabolic Activating Protein – basically a “transcription factor”. CAP needs to bind in order for the RNA Polymerase to bind. Cyclic-AMP activates CAP, causing an allosteric reaction so it can bind the promoter. , lactose present

XII. Gene Regulation The lac Operon in E. coli Within the promoter, there is a binding site for Catabolic Activating Protein – basically a “transcription factor”. CAP needs to bind in order for the RNA Polymerase to bind. Cyclic-AMP activates CAP, causing an allosteric reaction so it can bind the promoter. So, the binding of CAP stimulates transcription. , lactose present

XII. Gene Regulation The lac Operon in E. coli When Glucose is present, the concentration of c-AMP declines, it does not bind to CAP, and CAP does not bind to the Promoter; so the RNA Poly does not bind either and the genes are off. , lactose present

CAP REPRESSOR

XII. Gene Regulation The lac Operon in E. coli When Glucose is present, the concentration of c-AMP declines, it does not bind to CAP, and CAP does not bind to the Promoter; so the RNA Poly does not bind either and the genes are off. So, the lac operon is regulated first by the presence/absence of glucose; the needed nutrient…and then by the presence of lactose, which could be metabolized to produce glucose if necessary.

XII. Gene Regulation The lac Operon in E. coli B. The trp Operon in E. coli

XII. Gene Regulation The lac Operon in E. coli B. The trp Operon in E. coli Tryptophan is an amino acid that can be synthesized by tryptophan synthetase. This gene and its partners are only ON if tryptophan is absent. The presence of tryptophan represses the production of these enzymes (repressible system).

B. The trp Operon in E. coli

B. The trp Operon in E. coli ACTUALLY, TRANSCRIPTION ALWAYS PROCEEDS A LITTLE BIT…UP TO THE REGION CALLED THE “ATTENUATOR”…

B. The trp Operon in E. coli ACTUALLY, TRANSCRIPTION ALWAYS PROCEEDS A LITTLE BIT…UP TO THE REGION CALLED THE “ATTENUATOR”…

B. The trp Operon in E. coli ACTUALLY, TRANSCRIPTION ALWAYS PROCEEDS A LITTLE BIT…UP TO THE REGION CALLED THE “ATTENUATOR”…

B. The trp Operon in E. coli Two hairpin loops can form in the m-RNA; the 3-4 loop causes termination of transcription.

B. The trp Operon in E. coli Two hairpin loops can form in the m-RNA; the 3-4 loop causes termination of transcription. Because translation occurs as soon as m-RNA is produced, ribosomes jump on and begin to read the strand… there are two trp codons at the beginning of the sequence.

B. The trp Operon in E. coli Two hairpin loops can form in the m-RNA; the 3-4 loop causes termination of transcription. Because translation occurs as soon as m-RNA is produced, ribosomes jump on and begin to read the strand… there are two trp codons at the beginning of the sequence. If trp is present, the ribosome zooms along (incorporating trp) and it occupies the 2 region… region 3 is free to bind with 4 and the termination loop forms…

B. The trp Operon in E. coli Two hairpin loops can form in the m-RNA; the 3-4 loop causes termination of transcription. Because translation occurs as soon as m-RNA is produced, ribosomes jump on and begin to read the strand… there are two trp codons at the beginning of the sequence. If trp is present, the ribosome zooms along (incorporating trp) and it occupies the 2 region… region 3 is free to bind with 4 and the termination loop forms… If low trp, then ribosome stalls; region 3 bind to 2, no termination loop forms, and transcription of the genes proceeds… Translation of the genes begins at start codons downstream…

XII. Gene Regulation The lac Operon in E. coli B. The trp Operon in E. coli C. Regulation in Eukaryotes

XII. Gene Regulation The lac Operon in E. coli B. The trp Operon in E. coli C. Regulation in Eukaryotes - higher levels of packaging, intron-exon structure, and the need for tissue specialization makes regulation in eukaryotes far more complex than responding to environmental cues.

XII. Gene Regulation The lac Operon in E. coli B. The trp Operon in E. coli C. Regulation in Eukaryotes - higher levels of packaging, intron-exon structure, and the need for tissue specialization makes regulation in eukaryotes far more complex that responding to environmental cues. Histone Regulation - Core DNA, bound to histones, is OFF. Only “linker DNA”, between histones, is even accessible to RNA polymerases. So, binding DNA to histones is the first way to shut it off.

C. Regulation in Eukaryotes Histone Regulation - Three ways to reveal DNA “chromatin remodeling”

C. Regulation in Eukaryotes Histone Regulation - Three ways to reveal DNA “chromatin remodeling” Methylation - highly repetitive sequences - imprinted genes - Barr bodies

C. Regulation in Eukaryotes Histone Regulation - Three ways to reveal DNA “chromatin remodeling” Methylation - highly repetitive sequences - imprinted genes - Barr bodies Some proteins bind to the methylated cytosines, and may either recruit repressors or interrupt transcription factor binding.

C. Regulation in Eukaryotes Histone Regulation Methylation Promoters - Several consensus sequences (TATA, CAAT, GGGCGG) appear in combination in nearly all promoters and are required for basal levels of transcription

C. Regulation in Eukaryotes Histone Regulation Methylation Promoters Enhancers/Silencers Cis-acting elements on the same chromosome, which regulate a neighboring gene. They are somewhat like operators, in that they are binding sites for transcription factors that can “up” or “down” regulate transcription. However, they function ANYWHERE near the gene: before, within, or after

C. Regulation in Eukaryotes Histone Regulation Methylation Promoters Enhancers/Silencers Cis-acting elements on the same chromosome, which regulate a neighboring gene. They are somewhat like operators, in that they are binding sites for transcription factors that can “up” or “down” regulate transcription. However, they function ANYWHERE near the gene: before, within, or after They are not gene specific – they will enhance their neighbor Silencers tend to reduce binding of the polymerase to the promoter.

C. Regulation in Eukaryotes Histone Regulation Methylation Promoters Enhancers/Silencers These are the transcription factors that bind to enhancer and silencer regions of the human metallothionien IIA gene promoter region!! - What does having all these modifiers allow for?

C. Regulation in Eukaryotes Enhancers/Silencers Transcription Factors - These are the proteins that bind to DNA and influence transcription. They have “binding domains” that bind DNA in particular ways.

C. Regulation in Eukaryotes Enhancers/Silencers Transcription Factors - These are the proteins that bind to DNA and influence transcription. They have “binding domains” that bind DNA in particular ways. HTH = “helix-turn-helix” One class of important HTH TF’s contain specific sequences of AA’s called a homeodomain. This is encoded by a 180 bp region in it’s gene called a homeobox. These homeotic genes/proteins are conserved across all eukaryotes and are critical to basic animal development.

C. Regulation in Eukaryotes Enhancers/Silencers Transcription Factors - These are the proteins that bind to DNA and influence transcription. They have “binding domains” that bind DNA in particular ways. “Zinc-Finger”: Zinc binds to two cysteine and two histidine AA’s. The sequence between forms A loop or “finger”, and the specific AA sequence Binds specific DNA sequences…

C. Regulation in Eukaryotes Enhancers/Silencers Transcription Factors - These are the proteins that bind to DNA and influence transcription. They have “binding domains” that bind DNA in particular ways. bZIP=“basic leucine zipper”: leucine AA’s in Different chains dimerize and the leucines “zip” The other alpha-helices bind specific DNA sequences

C. Regulation in Eukaryotes Enhancers/Silencers Transcription Factors - These are the proteins that bind to DNA and influence transcription. They have “binding domains” that bind DNA in particular ways. - Then, the TF’s have other biding sites for Proteins (like basal transcription factors) or Other chemicals (like hormones)

C. Regulation in Eukaryotes Transcription Factors Alternate Splicing Pathways - Many proteins can be made from the same gene, by splicing the m-RNA differently. Humans have 20-30K genes, but several 100,000 proteins! A calcium regulator in the thyroid A hormone made in the brain

C. Regulation in Eukaryotes Alternate Splicing Pathways 7. Controlling m-RNA stability Existing tubulin units interact with a new tubulin strand and translation stalls, releasing RNAse that cleave the m-RNA. So tubulin is only made when free tubulin units are not present.

C. Regulation in Eukaryotes Controlling m-RNA stability RNA Silencing - Short pieces of RNA can bind to DNA in the nucleus or m-RNA in the cytoplasm and regulate gene expression.

C. Regulation in Eukaryotes Controlling m-RNA stability RNA Silencing - Short pieces of RNA can bind to DNA in the nucleus or m-RNA in the cytoplasm and regulate gene expression. - si-RNA: Initially they are found as ds-RNA (probably as the result of hairpin); they are cut by the ‘dicer’ protein into 21 base sequences (short-interfering RNA’s ) that bind a protein and, as ss-RNA, bind m-RNA and the protein cleaves the m-RNA.

C. Regulation in Eukaryotes Controlling m-RNA stability RNA Silencing - Short pieces of RNA can bind to DNA in the nucleus or m-RNA in the cytoplasm and regulate gene expression. - si-RNA: Initially they are found as ds-RNA (probably as the result of hairpin); they are cut by the ‘dicer’ protein into 21 base sequences (short-interfering RNA’s ) that bind a protein and, as ss-RNA, bind m-RNA and the protein cleaves the m-RNA. - miRNA (microRNA): quite similar, but as ss-RNA they bind m-RNA and just stop translation. They are involved in developmental regulation – many bind m-RNA for transcription factors. They also can bind DNA and induce methylation in promoters, turning genes off. This is all RNA silencing……