12 - Gene Regulation
Tumor-supressor genes
-Promote cancer when both copies are mutated (must be homozygous for the mutated trait). -Tumor-suppressor genes function like the repressor in the E. coli lactose operon. Cancers appear when their respective repressors do not function
Breast Cancer: BRCA1 gene is a tumor-supressor gene
BRCA1 gene must have two mutations for a person to develop breast cancer. this can happen in one lifetime to a woman with homozygous for the unmutated version, but it is rare. more commonly, heterozygous women inherit one mutation already, and a second happens in her lifetime, causing cancer. Healthy= BRCA1. Unhealthy=brca1
How Eukaryotes regulate suites/groups of genes at once
Eukaryotes do not have operons. But some pathways require many genes at once, so co-regulation of related genes is required. An enhancer for Gene A requires a specific set of activators, lets say activators 2, 5, 7, and 8. An enhancer for Gene B requires activators 1, 5, 8, and 11. Any gene that also has activator sites 2, 5, 7, and 8, will be turned on at the same time as Gene A is on. The same goes for Gene B and activators 1, 5, 8, and 11. So through the combination of many overlapping activators, the cell can isolate one specific gene or turn on whole sets of genes spread throughout the genome at the same time. For one gene, it is the interaction of all the various activators and repressors and their levels of abundance that decides whether and how fast it will be transcribed.
Transcriptional regulation example: lactose
If you eat dairy, you have lactose sugars in your intestines. So you need a specific enzyme to digest it (break it down). The E. coli in your intestines do not need to back that enzyme unless you have just eaten dairy, so there are blocks on its genome that prevent it from making that particular enzyme unless lactose is present in the cellular environment.
Promoter
The promoter is a regulatory sequence of DNA; it is the site to which RNA polymerase binds. It is the first regulatory sequence in the operon.
Structure of a Eukaryotic Gene
The promoter, immediately upstream of the transcription unit (the sequence that actually codes for a protein or RNA), contains sequence elements (such as a TATA box—looks like the -10 sequence in E. coli) that are important in transcription initiation. Transcription factors recognize and bind to the TATA box, and recruit RNA polymerase II (forming the RNA polymerase II-transcription factor complex). Just before the promoter is the promoter proximal region that contains binding sites for regulatory proteins
Transcriptional Regulation
Transcriptional regulation are the processes that directly control gene expression (which ones get transcribed, AND what modifications happen to the RNA after transcription, AND where it goes, AND whether it is translated, AND what happens to the protein or modified RNA after that). Every step of the process of gene expression can be regulated, and can be regulated in infinite ways (cutting, attachments, folding, etc.) *** Cells can VERY rapidly turn on and off different metabolic processes to respond to environment.
Natural defense against cancer
*No SINGLE mutation results in metastatic (spreading/dangerous) cancer. typically you need four or more things to go very wrong (somatic mutations) in order for dangerous cancer to happen.
Review of Operons
1. Clustered, contiguous structural genes 2. Expression of genes is coordinately controlled (all or none are expressed) 3. promoters and operators=DNA sequences that bind proteins that regulate RNA polymerase recruitment and activity. 4. activators and repressors = DNA-binding proteins that bing to promoters and operators that regulate RNA polymerase recruitment and activity. can speed up or prevent transcription. 5. Small effector molecules (e.g. Allolactose and cAMP in the case of the lac operon, trp in the case of the trp operon) are used to discern the needs of the cellular environment
Several Different levels of Gene Regulation in Eukaryotes
Eukaryotic cells are more complex: -Nuclear DNA is organized with histones into chromatin. -Multicellular eukaryotes produce many types of cells that need to use different genes. -The nuclear envelope separates transcription and translation. Transcription happens in nucleus, translation happened in ribosomes in the rough E.R., outside nucleus. Gene expression is regulated at many levels: Transcriptional regulation Posttranscriptional regulation Translational regulation Posttranslational regulation
Operator
Other regulatory sequences in the DNA. A regulatory protein binds onto DNA here.
Transcription Regulation of an Inducible Operon: Lac Operon
The lac operon is called inducible because it is off until lactose is present and then it is turned on. (Negative control= the lac repressor, which needs allolactose to release and allow the lac operon to activate). Lactose is turned into glucose to undergo glycolysis, but if glucose is present in good amounts the bacteria will not bother with the energy of using the lac operon; it will use the glucose first. SO, another level of control: if lactose is present, but glucose is also abundant, the cell will keep the lac operon turned off. This requires positive control.
Example: Colorectal cancer
What needs to happen: 1)activation of oncogene (a small benign tumor aka adenoma forms) 2)loss of tumor-supressor gene (a larger, significant but still benign adenoma forms) 3)loss of a second tumor suppressor gene (causes growth of malignant carcinoma that can spread) ***It takes a long time and many many divisions to get all three or more mutations, which is why you don't need to be checked for colon cancer until old age.
Example: Myc
Myc is an example of an proto-oncogene. The Myc gene codes for a transcription factor that influences the activity of 15% of human genes. If mutated to a hyper-active form, the effect is to increase transcription and accelerate cell division.
The 5' and 3' untranslated regions
There is a start site on DNA where transcription starts. This is not the exact same place as the AUG sequence on mRNA that is the start site of translation. So there is a portion near the beginning of the mRNA (the 5' end) that is transcribed but not translated. In the same way, there is a portion of nucleotides that get transcribed but not translated because they are after the stop codon in mRNA.
Transcription regulation in prokaryotes aka bacteria
Transcription regulation in prokaryotes involves operons. operons are not present in eukaryotes.
Example of regulation of multiple genes in eukaryotes: steroid hormone
A hormone is made in one area of the body and carried through the blood stream to another place in the body (the target tissue). A steroid hormone acts only on specific target tissues that have steroid hormone receptors in cytoplasm. A single steroid hormone regulates all genes in the entire genome that have the same regulator sequence. Plasma membranes keep out polar molecules and hydrophilic molecules. Steroid hormones are hydrophobic and small and non-polar; they cross plasma membrane very easily and reach the receptor in cytoplasm.
The Genetic Basis of Cancer
Cancer is caused by mutations in genes that control cell division. Mutations in two types of genes can cause cancer: Positive regulation of cell division. Proto-oncogenes normally promote cell division, but in a highly regulated fashion. Mutations to oncogenes enhance activity and make cells divide more. Tumor-suppressor genes: Negative repression of cell division. Normally inhibit cell division. Mutations can inactivate the genes and allow uncontrolled division to occur.
Transcription Regulation in Eukaryotes
Eukaryotes do not have operons. Each gene has its own separate promoter. Eukaryote genes are most commonly switched off and need activators to be turned on to be transcribed. (because eukaryote RNA polymerases are not very smart or good at binding to DNA, they need help finding and binding to the promoter, as opposed to bacterial RNA polymerase which is quite effective and site-specific and goes straight to the site where it is meant to be). There are whole suites of interrelated regulatory genes that interact in complex ways to turn on or off various suites of genes. Eukaryotes have short term and long term regulation. Short term is turning on and off sets of genes in response to stimuli. Long term is turning on and off sets of genes needed for growth and development of specialization. There are several different types of RNA polymerase in eukaryotes.
Positive vs. Negative Control
Gene regulation can be positive or negative. Positive Control: the gene is normally off; in its unaltered state it will not be transcribed. To induce it to activate, some new protein must be introduced that allows RNA polymerase to bond to the promoter of that gene and transcribe it. Negative Control: the gene is normally on; in its unaltered state it is always being transcribed. A regulatory protein must be attached to block it, and when it needs to be read that block falls off or is deactivated.
Activation of Transcription in Eukaryotic Genes
General transcription factors bind at/around the promoter/TATA box/promoter proximal region. Once they have bound to DNA they bind with RNA polymerase to make the transcription initiation complex. For some genes, not just the general transcription factors at the promoter must bind, but the enhancer, far away on the genome, must fold closer and bind w/ the other proteins at the promoter to activate the polymerase. It can simply fold over and have the activators at the enhancer interact with the activators at the promoter, or there can be a coactivator protein that sits in-between the two strand which each bond to it. *****The logic of activators and repressors, responding to cellular stimuli, inducible vs. repressible vs. uninducible vs. constitutive, all holds for both bacteria and eukaryotes.
Problem faced by Eukaryotes and not prokaryotes is the problem of distance of regulators along genome from the promoter
In bacteria, only a few elements can regulate a gene because the regulatory elements must be very close on the genome to the promoter in order for the proteins produced by them reach the promoter with accuracy. Eukaryotes regulate by committee; a bunch of elements of the system must be in agreement to activate a gene. Where is the space for the code for all these elements? The DNA folds up to bring elements further along the genome close to the binding sites of the proteins they produce. These are called the enhancers. They could be a long long way away from the gene they regulate, on either side, or inside the gene.
Operon
In bacteria/prokaryotes, genes that code for related functions/proteins are clustered together in an operon. An operon has one set of regulatory proteins that regulate multiple genes together, so the set of genes can be made at one time in one mRNA strand. Some operons could be controlled a repressor protein, which prevents that gene being expressed. Others could be controlled by an activator protein, that turns on that gene's expression. One operon may be controlled by more than one regulatory protein/mechanism, and one regulatory protein/mechanism may control more than one operon.
Oncogene
Oncogenes promote cancer when present in a single copy. Can be viral genes inserted into host chromosomes by phages. Or they can be mutated versions of proto-oncogenes, normal genes that promote cell division and differentiation. Converting a proto-oncogene to an oncogene can occur by: 1) Mutation causing increased protein activity. 2) Increased number of gene copies causing more protein to be produced. 3) Change in location putting the gene under control of new promoter, that might increase transcription or put tons of new proteins in a tissue they're not supposed to be in.
Transcription Regulation of a Repressible Operon
Synthesis of Tryptophan by E. Coli. E. Coli can synthesize tryptophan, but it won't synthesize it if you have just eaten it, because it can use the ingested tryptophan instead.. It is repressible as opposed to inducible because it is normally always working, unless something new is introduced (tryptophan) that makes it stop. The trp operon codes for 5 genes that all help make tryptophan. Expression of trp operon is negatively controlled by a regulator gene, trp R, that continuously makes an inactive repressor protein. so usually the gene is not repressed. the repressor protein must be activated by presence of tryptophan (the corepressor) to work. This is NEGATIVE control, because the regulatory molecules discourage transcription. ****You can ask the same question about repressible as you can about inducible. What happens if you break one part of the system? does it become constitutive, always repressed, etc.?
The levels of activation of eukarotyic genes
1)General transcription factors bind around promoter to recruit RNA polymerase and start transcription at a base rate. 2) Specific activators can bind to promoter proximal elements to speed up transcription. 3) Activators can bind to the enhancer regions to greatly speed up transcription (the fastest speed)
Review of how a proto-oncegene can mutate into an oncogene to cause cancer:
1)Mutation within the gene can turn it into an oncogene, leading to hyper-active growth 2)Multiple copies of the gene can be made accidentally, causing normal growth in each but overall excess of protein 3)Gene could be moved to new DNA locus under new controls, causing the gene to make protein in excess
review: ways of regulating cell division
1)proto-oncogenes stimulate cell division in the presence of a growth factor molecule. They allow protein to be made that is released from the cell telling nearby cells to divide. 2)tumor-supressor genes repress cell division in the presence of a growth-inhibiting factor. They allow protein to be made that tries to get out of cell to tell other cells to divide, but it forces it to be made incorrectly so it can't get out of cell membrane.
Why is gene regulation needed?
All the cells in your body contain the exact same genome in its entirety. But they are all different and perform different functions; not all genes are expressed in one cell at one time, only certain ones needed to perform its function. So we must be able to regulate which genes are expressed (transcribed) at a given moment.
Example: retinoblastoma (Rb) gene
An example of a tumor suppressor is the gene for retinoblastoma (Rb). Rb protein controls the G1-phase to S-phase transition in the cell division cycle. Heterozygous individuals that have one mutated copy and one normal copy of Rb can develop the disease when their normal copy undergoes mutation or gets left out of one cell division. This happens in the tissue of the eye, where tumors of the retina form. Rb is targeted (inactivated) by HPV E7 (so getting HPV can cause cancer because it inactivates Rb gene, so Rb cannot stop cell division).
Review of the gene regulation of Lac Operon
It is an inducible operon, meaning it is normally kept off unless something is introduced to the environment that triggers it. It is regulated by one negative and one positive control. The negative: The Lac I regulator sequence (a sequence of DNA separate from the operon) is continuously transcribed to produce lac repressor protein that binds to the operator sequence in the lac operon. If lactose is present, the very minimal amount of beta-galactosidase (there is always a tiny bit being transcribed) turns some of it into allolactose (the inducer metabolite), which binds with the repressor protein, making it undergo a conformational change so it can't bind to the operator sequence and so can no longer block the RNA polymerase from transcribing the lac operon. The positive: a regulatory protein, CAP, binds to the CAP binding site right at the beginning of the lac operon promoter. but in order to work, it needs cAMP to bind to it. cAMP is produced only if glucose is not present. When glucose is not present, cAMP is produced, it binds to CAP, and CAP is able to do its job of speeding up transcription of the lac operon. SO, in order for the lac operon to be transcribed at full capacity, there needs to be an absence of glucose (ensured by positive control) and a presence of lactose (ensured by negative control).
Structure of the Lac Operon
The Lac Operon goes: CAP site-Promoter-Operator-Lac Z-Lac Y-Lac A-Terminator Elsewhere in the genome, outside the Lac operon, there is a regulatory gene called the Lac I gene that encodes for the regulatory protein of the operon, called Lac repressor. If NO lactose is present, Lac I produces lac repressor, which binds to the operator sequence in the lac operon and stops RNA polymerase from binding to the promoter sequence (prevents transcription of the lac operon). This is negative control. If lactose IS present, an inducer (called allolactose) binds to the lac repressor and makes it undergo a conformational change and fall off of the operator, so that the promoter is free to bind to RNA polymerase (transcription is induced).
CAP and cAMP
The regulatory protein CAP (catabolite activator protein) is an activator protein that encourages RNA polymerase to transcribe the lac genes really fast. It is synthesized in inactive form (not yet able to do its job of making polymerase go fast). There is a CAP bonding site right before the lac promoter on the DNA. If glucose is ABSENT in cell, cAMP (cyclic AMP) binds to and allows the CAP to be active and encourage RNA polymerase to transcribe the the lac operon really fast. If glucose is present, cAMP is not made and CAP stays inactive, so it doesn't encourage polymerase to transcribe the lac operon. It is still transcribed a tiny bit, but very slowly, and makes only a tiny amount. This is positive control because the regulatory molecules involved encourage transcription. -cAMP is made by an enzyme called adenylate cyclase. It turns ATP into cAMP. When glucose enters the cell, adenylate cyclase activity decreases so there is less cAMP to bind with/activate the CAP and so RNA polymerase acts much slower over the lac operon, effectively halting lactose catabolism.
The 3 genes of the Lac Operon
Three different genes encode for enzymes that break down lactose (for the catabolism of lactose). They are combined together into an operon called the Lac Operon. The 3 adjacent genes: Lac Z, Lac Y, Lac A Lactose= a disaccaride (two sugar rings); one monomer of galactose, one monomer of glucose. Lac Z makes the first enzyme, beta-galactosidase, that cuts the two rings of the lactose apart into galactose and glucose. The galactose becomes a glucose. The two glucose molecules are then ready to begin glycolysis (have been successfully digested or catabolized). BUT, lactose is a disaccharide, a little too big to permeate the cell membrane on its own. Lac Y makes the enzyme Permease, that allows the lactose to get inside the cell so it can be digested. Lac A makes the enzyme transacetylase. The first two enzymes can accidentally bring substrates other than lactose into the cell, so this enzyme moves around acetyl groups on other substrates to deal with them or make sure they are ready to be cleaved by beta-galactosidase.
Other terms: un-inducible and constitutive
Un-inducible- you cannot turn the gene on Constitutive- you cannot turn the gene off Could be weakly inducible (some but not all) ***Possible quiz question: if you introduce a mutation that breaks one part of the system, what does the operon become? Does it stay inducible? become one of the above conditions?
Comparing lac and trp operons
lac: inducible, b/c the repressor is synthesized in active form, so without any alterations it is off. it needs another molecule to help it turn on. the inducer (allolactose) deactivates the repressor so operon can be transcribed. controlled by one negative (allolactose) and one positive (CAP and cAMP) control trp: repressible, because the repressor is synthesized in inactive form, so without any alterations it is on. it needs another molecule to help it turn off. the corepressor (tryptophan) activates the repressor so operon cannot be transcribed. controlled by one negative control (tryptophan)
