PROKARYOTIC GENE REGULATION

- prokaryotes can grow with amino acids, vitamins, nucleotides, carbohydrates, and certain temperatures
- prokaryotic genomes contain the needed information for adapting to environmental changes
- housekeeping genes must be expressed at all times for normal cellular function
- encodes for structural proteins, RNA and DNA polymerase, and ribosomes
- maintain general cellular activities when cell is responding to changes
- regulated genes are only expressed at certain times, under certain conditions  most genes
- expressed after an environmental cue
- codes for enzymes that change growth and cell division

REGULATION OF LACTOSE GENE

- expression of genes for enzymes will change when nutrient availability changes
- glucose is the preferred energy source for E. Coli
- bacteria will switch to metabolizing lactose after glucose is used up
- growth until glucose is consumed  no growth  growth as lactose is metabolized
- the lactose metabolizing enzyme is only translated when lactose is available and glucose isn’t

JACOB AND MONOD’S EXPERIMENT

- b-galactosidase cleaves lactose into glucose and galactose
- background: b-galactosidase is only observed when lactose is present
- hypothesis: without lactose, b-galactosidase is either not produced or degraded after production
- experiment: lactose is added and removed from a culture of E. Coli cells
- results: b-galactosidase levels increase when lactose is added, remain stable when lactose is removed
- conclusions: b-galactosidase gene is only expressed when lactose is present  regulated gene

LEVELS OF GENE REGULATION

- gene expression includes transcription, translation, and protein modification
- transcription control = can proteins bind to the promoter and produce mRNA
- translational control = does mRNA degrade, can ribosomes bind to mRNA, rate of translation
- post-translation control = how are proteins folded and modified
- if any step is disrupted, the functional protein is not produced

RATE OF GENE REGULATION

- post translational regulation is fastest
- inactive proteins can be stockpiled and rapidly activated after a signal
- transcriptional regulation is the slowest but most efficient
- transcription, translation, and modification must occur before protein is produced
- energy and resources are wasn’t making inactive protein stockpiles
- transcriptional regulation occurs after a major environmental change  e.g. no glucose available
- bacteria switch to metabolizing lactose if lactose is present and glucose isn’t
- beta galactosidase and lactose permease are only produced when lactose is metabolized
- lactose permease = transmembrane protein that moves lactose into the cell
- beta galactosidase = cytoplasmic enzyme that cleaves lactose into glucose and galactose
- lactose permease and beta galactosidase are both required for lactose metabolism

- in prokaryotes, groups of functionally related genes are organized into operons

- operon = promoter + operator + functionally related genes
- transcription can’t occur if a repressor protein is bonded to the operator
- an operon is transcribed into a single polycistronic mRNA molecule
- the polycistronic mRNA can code for multiple proteins  start and stop codons for each gene

- lac operon = promoter + lacO + lacY + lacZ

- the lacO gene is the operator
- lacY codes for lactose permease
- lacZ codes for beta galactosidase
- lacI is located near the lac operon, and encodes for a repressor protein

1. the lacI gene is continually expressed, producing the repressor protein  housekeeping gene
- the repressor protein is made of 4 identical protein subunits  tetrameric
2. the repressor protein binds to the lacO operator
3. the lac operon DNA twists into a loop
4. RNA polymerase can’t bind to the promoter, shutting off transcription
- glucose facilitates the expression of the repressor protein

- when lactose is present, the repressor can’t bind to the operator  inducer protein
1. lactose binds to specific binding sites on the repressor protein
2. lactose causes a conformational change in the repressor protein
3. the repressor protein detaches from the lacO operator
4. RNA polymerase can bind to the promoter, activating transcription

- adenylyl cyclase converts ATP into cyclic adenosine monophosphate (cAMP)

- adenylyl cyclase is inhibited by high glucose levels
1. low glucose levels cause an increase in cAMP levels
2. cAMP binds to a cAMP receptor protein (CRP), forming a CRP-cAMP complex
3. the CRP-cAMP complex binds to a CRP binding site on DNA
4. the CRP-cAMP complex recruits RNA polymerase to the promoter, activating transcription
- both glucose and lactose = low cAMP levels = no CRP-cAMP complexes = no transcription
- if the repressor is attached, the CRP-cAMP complex won’t activate transcription

- lac operon mRNA is highest when bacteria are actively metabolizing lactose
- when bacteria are metabolizing glucose, there are low, but never no lac operon mRNA
- the repressor protein will occasionally detach, leading to transcription of the lac operon
- the main energy source after birth is milk, which contains lactose
- lactase is found on the membranes of intestinal microvilli cells, facing the lumen
- lactase hydrolyzes lactose into glucose and galactose
- glucose gets absorbed into blood, where it can be used for cellular respiration
- galactokinases convert galactose into galactose-1-phosphate
- gal-1-uridyltransferase and UDP-galepimerase convert galactose-1-phosphate into glucose-1-phosphate
- glucose-1-phosphate can be used for cellular respiration

- lactase gene expression is high in infants  primary energy source is lactose

- in 65% of adults, lactase levels decrease  less lactose present = lactase gene not expressed
- more likely in Asian and African populations
- in 35% of adults, lactase levels remain high
- cattle domesticated = more lactose = mutation in MCM6 = lactase gene is expressed
- more likely in European, Middle Eastern, and North African populations

- individuals with lactose intolerance aren’t able to digest lactose

- due to mutation in LCT gene on chromosome 2
- mutation leads to misfolded lactase enzymes which don’t function
1. lactose is not absorbed in small intestines
2. lactose enters large intestines
3. bacteria break lactose down and produce gases, causing bloating
4. high lactose concentrations = water enters large intestines = diarrhea and dehydration

- due to mutation in GALT, GALE, GALK1 genes on chromosome 9

- type 1 = Gal-1-uridyltransferase, type 2 = galactokinase, type 3 = UDP-galepimerase
- individuals with galactosemia can’t process galactose
- galactose is toxic and causes brain damage, cataracts, kidney failure, and liver damage (jaundice)
- galactosemia is treated by omitting galactose from diet

- yogurt and cheese contain bacteria  Lactococcus, Lactobacillus, bifidobacteria

- bacteria convert lactose into glucose and galactose, producing lactic acid
- lactic acid causes milk to harden and curdle
- lactic acid kills pathogens

- dietary aid pills contain lactase, which can break down consumed lactose
1. microorganisms are grown in a chamber  Aspergillus fungi or Kluveromyces yeast
2. lactose is added to the chamber
3. microorganisms produce b-galactosidase, which is made into pills

- lactase can be attached to alginate beads to make lactose free milk

1. milk is passed through alginate beads
2. immobilized lactase hydrolyzes 40% of lactose in milk
3. more lactase is added into the milk
4. when milk is consumed, the lactase gets denatured
DIFFERENTIATION
- all human cells can from a single fertilized egg, which divides into embryonic stem cells
- embryonic stem cells can differentiate into 200 types of specialized cells
- most human cells have the same genome
1. cells communicate by sending molecular signals
2. different signal = different genes expressed = different proteome
3. different proteomes create specialized cells that may form into different tissues and organs

TRANSCRIPTION FACTORS
- transcription factors bind to specific DNA sequences based on structural and chemical complementarity
- there are 4 types of transcription factors based on binding motif
1. helix-loop-helix
2. helix-turn-helix
3. zinc finger  zinc is required to activate TFs
4. leucine zipper  2 helices interact with each other
- transcription factors have alpha helices that fit into DNA grooves  amino acids interact with bases
- gene expression is halted when transcription factors can’t bind to DNA

ACTIVATING TRANSCRIPTION
1. general transcription factors bind to the core promoter region
- TBD subunit of TFIID to the TATA box 25bp from the start site
- TFIIB to the BRE region 35bp from start site
2. activator proteins bind to enhancer regions far from the core promoter
3. DNA loops around to bring the enhancer and promoter regions together
4. mediator complexes connect the proteins in the enhancer and promoters
5. RNA polymerase can recognize the promoter and start transcription

REPRESSING TRANSCRIPTION
- silencer regions are located far upstream the promoter region
- repressor proteins bind to silencer regions
- repressor proteins disrupt transcription factor and mediator activity
- as a result, RNA polymerase can’t bind to DNA and transcription can’t occur
PROKARYOTIC VS EUKARYOTIC DNA
- in both: RNA polymerase binds to promoters, proteins activate or repress transcription
- a prokaryotic promoter controls many genes, a eukaryotic promoter controls 1 gene
- in eukaryotic DNA, 150bps are wound twice around 8 histone proteins, forming nucleosomes
- eukaryotic DNA must be unwound before transcription
- as a result, prokaryotic genes are “on” while eukaryotic genes are “off”

HISTONE MODIFICATION
- positive R groups in histone proteins interact with negative phosphates in DNA
1. an activator protein binds to an accessible enhancer site
2. activator proteins recruit a histone acetyltransferase (HAT) coactivator enzyme
3. HAT adds acetyl groups to lysine amino acids along the R groups of histone proteins
- methylation of lysine and arginine, or phosphorylation of serine and threonine can occur
4. modifications affect the positive charge on R groups
5. DNA is unwound, less tightly wound, or more tightly wound
- adding acetyl or 1 methyl group activates transcription
- adding 3 methyl groups represses transcription

DNA METHYLATION
- CpG island = string of cytosines and guanines near or in the promoter
1. methyl groups are added to cytosine bases in CpG islands
2. the shape of the DNA binding site changes
3. RNA polymerase and TFs can’t bind to the promoter, halting transcription
4. histone deacetylase binds to DNA and deacetylates histones, compacting DNA
- methylation state is affected by environmental and developmental cues
- methylation is an epigenetic mechanism  affects gene expression without changing sequence
- methylation state can be inherited by daughter cell and offspring

HEMOGLOBIN
- blood cell progenitors produce hemoglobin before differentiating into red blood cells
- fetal hemoglobin = 2 alpha globin + 2 gamma globin
- adult hemoglobin = 2 alpha globin + 2 beta globin
- chromatin is coiled around beta globin gene in fetuses, gamma globin gene in adults
- switch from gamma to beta globin is regulated by TFs
- gamma globin has a higher affinity for oxygen
MICRO RNA
1. miRNA is transcribed from protein encoding genes
2. miRNA forms hairpin loop structures in the nucleus
3. cytoplasmic enzymes remove the loop and cut the stem into 20-25bp fragments
4. one strand of a miRNA fragment joins to an RNA-inducing silencing complex (RISC complex)
5. miRNA binds to target mRNA in a non-exact manner  degrades many types of RNA
6. proteins in the RISC complex block translation

SMALL INTERFERING RNA

1. siRNAs are transcribed and processed similarly to microRNAs
2. siRNAs join to a RISC complex
3. siRNAs bind to mRNAs in an exact manner  degrades one type of RNA
4. siRNAs cleave and destabilize the target mRNA

POST TRANSLATIONAL REGULATION

- post translational modifications are used to activate or inactivate proteins
- proteins are inactivated via selective degradation when they are no longer needed
1. target proteins are tagged with a ubiquitin chain
- involves activation, conjugation, ligation reactions using ATP
2. the target protein is passed through a proteasome protein complex using ATP
3. the proteasome unfolds and cleaves the protein into amino acids
- ubiquitin proteins are destroyed by other ubiquitin proteins
IN SITU HYBRIDIZATION
- used to study the expression of one or a few genes in living tissue samples
- if the target mRNA is detectable, the target gene is being expressed
1. the target gene produces a specific mRNA sequence
2. fluorescent single stranded DNA/RNA probes are manufactured
3. the fluorescent probe is injected into the tissue sample
4. the fluorescent probe binds to the target mRNA
5. fluorescent regions are expressing the target gene

MICROARRAY ANALYSIS
- microarray chip = glass slide with wells that each contain a gene of interest  can contain all genes
1. sample of cells is taken from patient and grown in a culture
2. mRNA is isolated from the cells
3. mRNA is mixed with reverse transcriptase enzymes to create fluorescent cDNA molecules
4. fluorescent cDNA molecules bind to the well that contains their gene
5. differences in colour of wells are measured with a scanner
- active gene = mRNA produced = cDNA produced = well for that gene is fluorescent

CANCER APPLICATION
- used to compare gene expression between normal and cancerous cells
- normal cDNA is labelled green, cancerous cDNA is labelled red
- green well = gene is upregulated in normal cell
- red well = gene is upregulated in cancer cell
- yellow well = gene is equally expressed in normal and cancerous cell
- e.g. red well for movement genes, green well for apoptosis genes, yellow well for ribosome genes

OTHER APPLICATIONS
- used to examine the expression of thousands of genes  are specific genes transcribed
- used to investigate whether transcription in groups of genes is coordinated
- used to compare gene expression between different stages of development, cell types, and signals
DETECTING A SIGNAL
1. a signalling molecule is sent to a target cell
2. receptors bind to the signalling molecule
3. a signal transduction cascade leads to short or long term responses
- short term = post translational modifications that modify cellular processes
- long term = changes in gene expression and development

EPIGENETICS
- chemical modifications that alter gene expression without altering the DNA sequence
- includes histone modifications and DNA methylation
- epigenetics change through human development
- epigenetics are influenced by diet, toxins, environmental factors, stress
- epigenetics are involved in diseases
- low or high levels of methylation = abnormal gene expression = cancerous cells
- e.g. apoptosis gene may be methylated
- more methyl-rich folic acid in diet = more methylation = lower risk of spina bifida

AGOUTI GENE
- the agouti gene makes proteins that cause brown mice to turn yellow
- agouti, obesity, diabetes, and cancer genes are off by default
- no methylation = genes are expressed = mice are yellow, obese, diabetic, and cancerous
- if mice are given diets with more methyl, offspring are more likely to be brown and healthy
- therefore, methylation affects gene expression, and is affected by maternal diet

TWIN STUDIES
- monozygotic twins have identical genomes  blastocyst collapse splits stem cells
- DNA methylation and histone acetylation was examined in 40 twin pairs from Spain, ranging from 3-74
- 3 year old twins had similar methylation patterns at the promoters
- 50 year old twins had different methylation patterns
- as a result, 50 year old twins had different gene expression patterns
- different conditions = different methylation = different gene expression = different characteristics
- e.g. smoking leads to more skin sagging, less face fat
- Dr. Parminder Raina is studying how epigenetics are affected by various factors