Unit 3 (Week 10 Gene Regulation) Flashcards
What is the gene function either at the level of traits or at the molecular level?
Gene expression
What is the ability of cells to control the expression of their genes?
Gene regulation
What are unregulated genes that has a relatively constant level of expression in all conditions over time?
Constitutive genes
What are the benefits of gene regulation? (2)
- Conserves energy. Proteins that are encoded by genes are produced only when needed.
- In multicellular organisms, gene regulation also ensures that genes are expressed in the appropriate cell types and at the correct stage of development.
What does E. coli do within gene regulation that pertains to its ability to uptake lactose?
When lactose is not present in the environment, an E. coli cell makes very little of these proteins, lactose permease (transporter) and B-galactosidase (breaks linkage in lactose and galactose). However, when lactose becomes available, the bacterium produces many more of these proteins, enabling it to readily use lactose from its environment.
What are one of the most amazing examples of gene regulation in humans?
Cell differentiation. The process by which cells become specialized into particular types. Cells may differentiate into muscle cells, neurons, skin cells, or other types.
They have the same genome but a different proteome. Or they may have the same genome and proteome but produce the same proteins at different rates.
How does gene regulation underlie the different morphologies of the various types of cells?
Gene regulation causes each type of cell to express its own unique set of proteins, which, in turn, are largely responsible for the morphology and function of that type of cell.
What is the protein responsible for delivering oxygen to the cell’s of a mammal’s body?
Hemoglobin
Genes that encode slightly different globin polypeptides are turned off and on throughout the embryonic stage of development and the fetal stage of development and to include at birth.
The differences between these proteins that are produced is their affinity to oxygen.
Where does gene regulation happen most commonly in bacteria?
In bacteria, gene regulation most commonly occurs at the level of transcription, which means that bacteria regulate how much mRNA is made from genes. When geneticists say a gene is “turned off,” they mean that very little or no mRNA is made from that gene, whereas a gene that is “turned on” is transcribed into mRNA.
Bacteria can also regulate genes is by controlling mRNA translation, and can be regulated post-translational level in which the protein’s function is controlled after it is synthesized.
What protein, in most cases, is needed for the regulation of transcription?
REGULATORY TRANSCRIPTION FACTORS which is a protein that binds to DNA, usually in the vicinity of a promoter, and affects the rate of transcription of one or nearby genes. (Repressors and Activators induce negative and positive controls on rate of transcription)
The REGULATORY SEQUENCE in a DNA sequence that functions as a binding site for REGULATORY TRANSCRIPTION FACTOR proteins, which influences the rate of transcription.
What is a transcription factor that binds to DNA and inhibits transcription?
Repressor
What type of control in transcriptional regulation happens by repressor proteins?
Negative control
What is a transcription factor that binds to DNA and increases the rate of transcription?
Activators
What type of control in transcriptional regulation happens by activator proteins?
Positive control
What is a molecule that affects gene transcription by binding to a regulatory transcription factor, causing a conformational change in that protein?
Small effector molecule.
These changes can determine whether or not a regulatory protein can bind to the regulatory sequence.
What are the two domains (sites) on activators and repressors?
One site where it allows them to bind to DNA and another site where small effecter molecules bind.
In bacteria, a set of two or more genes may be under the transcriptional control of a single promoter. What is this arrangement called?
An operon.
What is a group of genes are transcribed as a single unit, resulting in the production of _______ that is capable of encoding more than one protein?
Polycistronic mRNA
What is the advantage of a bacterium to have an operon?
To regulate a group of genes that encode proteins whose functions are used for a common pathway.
The genome of E. coli carries an operon, called the lac operon, that contains the genes to metabolize what?
Lactose
[Focus on lac operon]
What is transcripted from the lac operon?
What does the lac operon genes consist of? Be specific.
- A polycistronic mRNA that encodes multiple proteins for the utilization of lactose.
- The lac operon consists of a promoter (lacP), and three protein-encoding genes.
LacZ gene encodes B-galactosidase which is an enzyme that breaks down lactose into glucose and galactose. As a side reaction, it is also responsible for a side reaction of breaking down a small amount of lactose into allolactose. This is a lactose analogue meaning structually differernt but similar function.
LacY gene encodes lactose permease, which is the transmembrane protein needed to transport lactose into the cytoplasm of E. coli.
LacA gene encodes glactoside transacetylase, which covalently modifies lactose and lactose analogues (allolactose) by attaching an acetyl group (-COCH3). This attachment of aceytal groups to nonmetabolizable lactose analogues prevents their toxic buildup.
Which genes are under the control of the lac promoter?
The lacZ, lacY, and lacA genes are under the control of the lac promoter.
What “sandwhiches” the lac promotor or lacP in the lac operon and are condisered regulatory sequences?
Note: Regulatory sequences are the areas that repressors and activators attach to.
On the left, we have the CAP site, which is a site recognized by the catabolite activator protein (CAP).
On the right of the promotor, we have the operator or lacO, which is a sequence of bases which provides a binding site for a repressor protein.
What is adjacent to the lac operon?
The lac regulatory gene. This gene, known as lacI, encodes the lac repressor and has its own promoter called an I promoter.
This repressor protein is important for the regulation of the lac operon. The lacI gene, which is constitutively expressed at a fairly low level, has its own promoter called the i promoter. The lacI gene is not considered a part of the lac operon.
What did Monod and Jacob do, under the interest of enzyme adaption, which occurs when a particular enzyme appears within a living cell only after the cell has been exposed to the substrate for that enzyme?
They exposed bacteria to lactose, the levels of lactose-utilizing enzymes in the cells increased by 1,000- to 10,000-fold. After lactose was removed, the synthesis of the enzymes abruptly stopped.
The first mechanism Jacob and Monod discovered involved the lac repressor, which binds to the sequence of bases found at the lac operator site, which prevented RNA polymerase from what?
Note: The lac repressor is synthesized by the expression of the lac regulatory gene i.e. the lacI segment.
From transcribing the lacZ, lacY, and lacA genes.
T/F RNA polymerase can still bind to the lacP (promotor) when the lac repressor is bound to the operator site.
True but the polymerase cannot move past the operator to transcribe lacZ, lacY, and lacA genes.
Note: With the lac repressor bound to the lacO site, and polymerase not able to move past it means that the lacO site or operator site lies after the promoter site, or lacP site.
What depends on whether or not the lac repressor binds to the operator site?
Allolactose. This is a product of B-galactosidase enzyme, a side reaction or conversion of lactose when it is present.
This is because allolactose is a small effector molecule and it adheres to the four subunits within the lac repressor which THEN induces a conformational change and prevents the repressor from binding to the operator.
RNA polymerase is free to transcribe the lac operon.
What is the importance of E. coli and the regulation of the lac operon?
It conserves energy since the proteins that utilize lactose are made only when lactose is present in the environment.
The respressor protein, lacI, is made in small quantities when lactose is not present which prevents the lac operon to express. Since allolactose is created by B-galactosidase when lactose is present, it effectively creates a conformational change in lacI therefore increasing the rate of transcription (without an activator). What is allolactose called?
In this case, what can the lac operon be called because of the effects of the inducer?
An inducer which is a small effector molecule that increases the rate of transcription.
The lac operon is an inducible operon because transcription increases which the presence of a small effector molecule.
What is the reality of a repressor and the proteins of the lac operon?
The respressor does not 100% repress the lac operon. The amount of B-galactosidase, lactose permease, and galactoside transaceytalase is very small but is present for when lactose shows up in the environment to jump start the process of metabolism.
The presence of lactose increases the cytoplasmic levels of allolactose gradually and eventually increases the transcription of the lac operon.
With regard to regulatory proteins and small effector molecules, explain the meaning of negative control and inducible.
Negative control refers to the action of a repressor protein, which inhibits transcription when it binds to the DNA. Inducible refers to the action of a small effector molecule. When it is present, it promotes transcription.
Who were the pioneers who focused on mutated strains of E. coli, specifically the mutation of lacI (repressor DNA), where lactose enzymes were created without the presence of lactose?
The study of merozygotes, bacterial cells containing F’ factor, or F prime factors. This study allowed the scientists exchange circular segments of wild type DNA to bacteria (E. coli) that had DNA which was mutated.
Jacob, Monod, and Pardee
When they added normal genes to the cell of mutant genes, the regulation of the mutant gene, meaning its constitutive expression of lacZ, lacY, and lacZ proteins, was shut off as well. This helped them understand the function of the lacI- mutation since the merozygote was able to introduce lacI+ (normal functioning lacI repressor gene) which inhibited the lac operon from creating proteins for both the gene with the mutation and the one without.
What were the three genetic terms to come from Jacob, Monod, and Pardee’s experiment with mutant lacI genes in E. coli?
Cis-acting element - A DNA segment that must be adjacent to the gene(s) that it regulates. Ex. The operator site (lacO) is a cis-acting element for the lac operon.
Trans-effect - A form of genetic regulation that can occur even though two DNA segments are not physically adjacent. Ex. The action of the lac repressor on the lac operon is a trans-effect.
A cis-effect is mediated by a cis-acting element that binds regulatory proteins, whereas a trans-effect is mediated by genes that encode diffusible regulatory proteins. Diffusible means it can spread through the cytoplasm.
[Focus on Activator for lac operon] 1. Even though the lac operon is under negative control by a repressor, it is also positively controlled by what?
- What is the small effector molecule that controls the CAP?
- When influenced by CAP and cAMP, what type of control is this?
- When cAMP binds to CAP, what transpires next?
- The CAP (catabolite activator protein)
- The cyclic AMP (cAMP) which is produced from ATP via an enzyme known as adenylyl cyclase.
- Positive
- Well, the cAMP-CAP complex then binds to the CAP site near the lac promoter, which causes the DNA to bend that enhances the ability of RNA polymerase to bind to the promoter. Transcription is then increased.
What is the key functional role of CAP and what does it cause?
Allows E. coli to choose between different sugars as an energy source.
It causes catabolite repression, meaning the prescense of a preferred energy source inhibits the use of other energy sources.
EXAMPLE
In the case of transcription, the presence of glucose (which is a catabolite; it is broken down-catabolized-inside the cell) causes catabolite repression and decreases rate of transcription by inhibiting the production of cAMP, thereby preventing the binding of CAP to the DNA.
Glucose blocks activation of the lac operon.
Sounds wierd? CAP and its relationship with increasing synthesis of operon proteins? Name was chosen before understanding that CAP actually increases the rate of anabolic properties.
What is the function of cAMP in eukaryotic cells?
In eukaryotic cells, cAMP acts as a second messenger in signal transduction pathways.
[Four possible environmental conditions that E. coli might experience with two sugars]
- High lactose, high glucose - What happens?
- High lactose, low glucose - What happens?
- Low lactose, high glucose - What happens?
- Low lactose, low glucose - What happens?
- CAP does NOT bind, but low level of transcription does occur. Repressor protein is inactive, and E. coli uses glucose.
- CAP does bind (no catabolite repression) to CAP site, lac repressor is inactive due to allolactose, meaning lac operon very high transcription.
- CAP does NOT bind, lac repressor binds as well blocking RNA polymerase. Very low rate of transcription.
- Regardless, when lactose is low, the lacI gene is expressed without allolactose meaning the lac repressor gene represses transcription of the lac operon even if glucose is low.
What are the advantages of having both an activator and a repressor protein?
In the case of bacterial metabolism of sugars, the repressor keeps the lac operon turned off unless lactose is present in the environment. The activator allows the bacterium to choose between glucose and lactose.
[Focus on operon that does biosynthesis in E. coli]
- What is an example of an operon in E. coli that involves the encoding of enzymes involved in biosynthesis and not the breakdown of a molecule?
- What does this operon make?
- What genes does this polycistronic mRNA express specifically? (5) These enzymes are involved in the pathway where its end product is tryptophan.
- What is the gene that regulates the trp operon via a repressor protein?
- Guess where the trpR binds to for regulation?
- How is the trpR controlled?
- With the answer to 6, what do you know about this whole process?
- The trp operon
- It encodes the enzymes needed to make the amino acid, tryptophan, a building block of proteins
- trpE, trpD, trpC, trpB, and trpA.
- trpR
- The trp operon operator site.
- It is controlled by the presence of tryptophan, which is the product of the metabolic pathway.
- When tryptophan presence is low, trpR does not bind to the operator site, therefore allowing the cell to express trp operon which then readily makes tryptophan. When tryptophan levels are high, tryptophan acts as a small effector molecule and binds to the trpR repressor protein. This repressor undergoes a conformational change that then binds to the trpO or operator site inhibiting transcription of the trp operon.
So with the lac operon, the small effector molecule is allolactose increases the rate of transcription when lactose is present and it is known as an inducer. However, in the case of the trp operon, the small effector molecule is the product of trp operon transcription, which reduces transcription of the trp operon which sufficient levels of tryptophan exist. What do you call this small effector molecule in this case? And since this is NOT an inducible operon, what type of operon is it?
Tryptophan, as a small effector molecule, is called an co-repressor. The trp operon is a repressible operon.
The lac operon is categorized as an inducible operon because allolactose, its small effector molecule, induces transcription. By comparison, the trp operon is considered to be a repressible operon because its small effector molecule, namely tryptophan, represses transcription.
[Start transcription within Eukaryotes]
- What are the similarities with bacteria in transcription?
- What are the differences with bacteria in transcription?
- Activator and repressor proteins are used affecting the ability of RNA polymerase. Many eukaryotic genes are influenced by small effector molecules.
- Many genes are almost always organized individually, not as operons. Eukaryotic gene regulation is much more intricate, because of eukaryotic complexity.
What is the phenomenon wherehy a combination of many factors determines the expression of any given gene in eukaryotes?
Combinatorial control
For initial transcription in eukaryotic protein-encoding genes, what are the three features common among most promoters?
Regulatory elements, a TATA box, and a transcriptional start site.
[Focus factors that contribute to combinatorial control in transcription]
- The stimulation and inhibition of RNA polymerase is affected by what?
- What refers to the binding of small effector molecules, protein-protein interactions, and covalent bonds?
- What is necessary for the change in chromatin for RNA polymerase to easily identify a gene to be recognized and transcribed?
- What inhibits transcription, either by preventing the binding of an activator or by recruiting proteins that inhibit transcription?
All of these can play a part simultaneously, or maybe just two of them. Most cases, transcriptional regulation in eukaryotes involves the initiation at the promoter.
- One or more activators and repressors.
- The function of activators and repressors are modulated and changed.
- Activators
- DNA methylation
[Focus promoter of a eukaryotic gene]
- What does the TATA box and transcriptional site form?
- Where does transcription actually begin in DNA?
- What is the sequence, to include its primes, of the TATA box which is 25 bp before the transcriptional start site?
- What does the TATA box determine?
- What type of transcription happens when the core promoter acts alone?
- The core promoter.
- The transcriptional start site
- 5’-TATAAA-3’ sequence
- The precise starting point for transcription
- Basal transcription
The regulatory elements or sequences are DNA segments that regulate eukaryotic genes and are recognized by regulatory transcription factors that affect RNA polymerase to transcript at the core promoter. What are the two elements that either enhance the rate of transcription or prevent transcription of a given gene when its expression is not needed?
Bonus: How far can regulatory elements locations be found?
Enhancers & Silencers
Between 50-100 bp before the transcriptional site or core promoter. In some cases, over 10,000 bps away!!!
What RNA is responsible for transcribing protein-encoding genes?
RNA polymerase II
What are the three types of proteins that play a role in initiating transcription at the core promoter of protein-encoding genes?
Which two of these types of proteins must come together at the TATA box fo the core promoter so transcription can be initiated?
What does the two types of proteins form when they come together at the TATA box?
- RNA polymerase II
- 5 different proteins called general transcription factors (GTFs)
- A large protein complex called mediator
RNA polymerase II and the GTFs
Preinitiation Complex
What is the protein complex that partially wraps around the RNA polymerase II and the GTFs?
The mediator
Mediator derives its name from the observation that it mediates interactions between the preinitiation complex and regulatory transcription factors such as activators or repressors that bind to enhancers or silencers. The function of mediator is to control the rate at which RNA polymerase begins to transcribe RNA at the transcriptional start site.
In eukaryotes, what do the activators and repressors bind to?
The enhancers and silencers, respectively, and regulate the rate of transcription of genes.
They can regulate the function of RNA polyermerase by binding to GTFs or mediator (assuming that the enhancers and silencers are located there).
The binding of activators to an enhancers or repressors binding to silencers has what effect on the ability for GTFs and RNA polymerase to create the preinitiation complex?
The GTF known as transcription factor II D (TFIID) which functions to recognize the TATAbox and begin the assembly process. Comparatively the activators and repressors either enhance or reduce the effectiveness for TFIID to recognize the TATA box and therefore effects the binding of RNA polymerase II and other GTFs at the core promoter (if acting alone).
[Focus of the Mediator and regulatory transcription factors]
- What is something an activator interacts with that is a protein that increases the rate of transcription but does not directly bind to the DNA itself?
- What does the activator-coactivator complex stimulate the function of which causes RNA polymerase II to proceed to the elongation phase of transcription more quickly?
- What about a repressor?
- We have talked about two ways that regulatory transcription factors influence transcription. What is the third way?
- The coactivator
- The function of the mediator
- The repressor does the opposite and prevents RNA polymerase II from progressing to the elongation phase.
- Recruiting proteins that affect chromatin structure in the promoter region.
[Focus in Chromatin Structure and DNA Methylation]
- What is DNA associated with like proteins to create a structure called and what does it make up?
- For GTFs and RNA polymerase II to be able to transcript chromatin, what must happen?
- What involves the attachment of methyl groups to cytosine bases which also affects the chromatin conformation and gene expression?
- Chromatin and it makes up eukaryotic chromosomes.
- Chromatin must be in open conformation and not closed conformation which makes transcription difficult or impossible.
- DNA methylation
