Flashcards in Gene Regulation Deck (100):
What 3 mechanisms do cells use to regulate metabolic flow?
1.) By changing substrate concentration availability
2.) By changing the relative activity or efficiency of enzymes.
3.) By changing the quantity of enzyme present (The regulation of gene expression is important here.)
How is regulation of gene expression illustrated in differentiation?
By changing the quantity of an enzyme present, the behaviors of liver cells, brain cells, and skin cells all differ even though they all contain the same genetic makeup.
How does substrate availability impact enzyme activity?
1.) For many enzymes, affinity for a substrate (Km) is close to physiological concentration so the rate of utilization is responsive to changes in concentration.
2.) The availability of metal ions and cofactors may impact enzyme activity.
3.) The enzyme is affected by immediate environment.
4.) Enzymes are often compartmentalized and found only in certain organelles.
5.) Sometimes enzymes that catalyze a sequence of reactions are organized as part of a macromolecular complex.
What two mechanisms are used to alter enzyme activity?
Allosteric Regulation and Covalent Modification
What is Allosteric Regulation?
What is an example of Allosteric Regulation?
1.) The catalytic activity of regulatory enzymes is modulated by allosteric effectors. The effector binds to an allosteric (regulatory) site on the enzyme and causes a conformational change that impacts the catalytic activity of the enzyme.
2.) Negative feedback = End products block pathway.
What is Covalent Modification?
Interconvertible enzymes exist in 2 forms, active and inactive, depending on whether or not the enzyme has been covalently modified by the attachment a some group. Enzymes are either activated or deactivated by phosphorylation.
What Covalent Modification involves Activation by Proteolysis?
Some enzymes and hormones are initially synthesized in an inactive form. Protease action then removes part of the molecule to produce the active form.
Proinsulin --> Insulin, Protrypsin --> Trypsin,
(Remember: Proteolysis is permanent.)
In what 3 ways do prokaryotes regulate enzyme levels by controlling their rate of synthesis?
1.) Constitutive enzymes are made in fixed amounts. They are required in fairly constant amounts at all times and are often referred to as "Housekeeping genes."
2.) Some enzymes are only useful for the catabolism of certain nutrients only when that nutrient is present. These enzymes are made at only a low basal level. (Ex. Galactosidase is only made when lactose is present.)
3.) Some enzymes are used for biosynthesis of metabolites (amino acids) and are made when the end product is not present. If the end product is available, the synthesis of these enzymes is repressed. (Ex. Enzymes involved in tryptophan biosynthesis.)
What is the Lactose (Lac) Operon of E. coli?
The system consists of 3 enzymes: galactosidase, galactoside permease, and galactoside acetylase. When E. coli is growing where glucose is present, these enzymes are only at low level. When glucose and lactose are present, the enzymes are still at low level. When only lactose is present, they are all expressed in equal amounts (either high or low). The structural genes (coding for the amino acid sequence of the enzymes) are physically associated with regulatory genes to form the operon.
How is the Lac Operon of E. coli an example of Induction?
1.) In glucose, repressor binds to operator to block transcription. (This is negative control.)
2.) In lactose, the inducer binds to the repressor to release it from the operator and allow the RNA polymerase to proceed. Since glucose is low, cAMP will be high. cAMP binds to the CAP protein which then binds the CAP binding site, helping the RNA polymerase bind the promoter. (This is positive control.) The 3 enzymes are produced from one long polycistronic message (ZYA).
3.) In both, the inducer releases the repressor from the operator, but since glucose is present, cAMP will be low. With no cAMP-CAP complex, RNA polymerase will not be able to bind. (The effect of glucose is catabolite repression.)
How is the Jacob and Monod Model an example of Repression-Derepression?
Regulation by enzyme repression and derepression is usually used in anabolic pathways in which presence of the end product represses the enzyme and absence of the end product derepresses the enzyme.
(The tryptophan biosynthesis pathway is regulated in this way.)
How does the Jacob and Monod Model differ from the Lac Operon?
In the Jacob and Monod Model, the repressor is always present but is inactive (has no affinity for the operator) unless it is complexed with the corepressor (end product). Thus, if the end product is not available, the repressor does not bind to the operator and the enzymes are derepressed and are all made from a polycistronic message (ABC). If the end product is available, the repressor-corepressor complex binds to the operator and inhibits transcription by blocking the RNA polymerase after it binds the promoter site.
What is Attenuation?
Attenuation is regulation by premature termination of mRNA synthesis.
At what level is regulation occurring in the induction or repression of the Jacob-Monod Model and in Attenuation?
In the Jacob-Monod Model, regulation is at variable initiation. In Attenuation, regulation occurs at variable termination. In both cases. regulation is at the transcriptional level.
What is the mechanism of action for Attenuation?
In high amino acid concentrations, mRNA synthesis is terminated prematurely so that the biosynthetic enzymes are not made. In low amino acid concentration, mRNA synthesis is completed so that the biosynthetic enzymes are made. (This is coordinate expression.)
In Attenuation, what determines whether the mRNA transcript is terminated early or completed?
Whether the mRNA transcript is terminated early or completed depends on which of the several possible secondary structures form at the 5' end of nascent mRNA. The formation of the secondary structure depends on the rate of movement of the first ribosome to translate the message.
In Attenuation, what is the function of the leader peptide?
The 5' ends of these mRNA encode a short polypeptide called a leader peptide that contains seven codons for the amino acid (Histidine) being controlled by the operon. If Histidine is plentiful, ribosomes translate this region quickly, which results in a long leader peptide and formation of an attenuator stem between 3 and 4 that codes for stop. If Histidine is depleted, the ribosomes stall during translation and the attenuator stem forms between 2 and 3 and a polycistronic message signals transcription to continue.
What type of DNA sequence is found in prokaryotes?
Prokaryotes have a single copy of unique DNA.
What type of DNA sequence is found in eukaryotes?
Eukaryotes have a single copy (one copy per haploid genome, genes for most enzymes) that is either mostly reiterated (several to hundreds of copies of rRNA genes) or highly reiterated (thousands to millions of copies of human Alu sequences).
What are the 2 types of chromatin?
Heterochromatin = condensed, inactive
Euchromatin = open structure, transcriptionally active, subject to DNase I cutting
What are the definitions of Constitutive, Induction, Repression-Derepression, and Attenuation?
Constitutive - Always needed so always present (Housekeeping genes)
Induction - Turned on in response to environment (Lac operon)
Repression-Derepression - Turned off by presence of molecule then turned back on in absence of molecule
Attenuation - Determines premature termination of mRNA synthesis
What is the difference between prokaryotes and eukaryotes in reference to DNA size?
Humans have 1000 times more DNA but only 5 times more genes so differences are in gene regulation.
What is the difference between prokaryotes and eukaryotes in reference to where DNA is made and how it is packaged?
E. coli make their DNA in chromosome and plasmid then package it as supercoiled DNA in a circular genome.
Humans make their DNA in nucleus and mitochondria then package it as linear nuclear DNA in chromatin.
What is the difference between prokaryotes and eukaryotes in reference to where genes are found, how they are regulated and by what kind of message?
E. coli has genes in operons and regulates them based on what is in the environment using a polycistronic (all genes in a row) message.
Human genes are usually scattered (sometimes in families) and regulate them based on what is in the environment as well as stage of differentiation using monocistronic (not clustered) messages.
What is the difference between prokaryotes in reference to presence of introns, usefulness of DNA, and gene copies?
E. coli does not have introns but most of its DNA is useful and genes are mostly in single copy.
Humans have introns but there is a lot of useless DNA and genes are in a reiterated single copy.
How is Hemoglobin an example of how eukaryotic genes are sometimes organized into families?
Different Hb genes expressed during different stages of development: Zeta2Epsilon2 in embryo, Alpha2Gamma2 in fetus, Alpha2Beta2 + Alpha2Delta2 in Adult
Alpha, and Zeta genes are on chromosome 16.
Beta, Delta, Gamma, and Epsilon genes on chromosome 11.
What are 2 examples of PoC 1: Specific chromosomes or regions may be inactivated during development?
1.) One X chromosome in each female mammalian cell is inactivated by condensation to heterochromatin (Lyon hypothesis, Barr bodies, Calico cats).
2.) DNA methylation turns off genes.
What is an example of PoC 2: Specific chromosomal regions may be activated by translocation?
Genes for the variable and constant regions of antibody molecules may be joined by translocation before they are activated.
What is an example of PoC 3: Specific chromosomal material may be lost during development?
The human red blood cell loses its entire nucleus.
What is an example of PoC 4: Gene reiteration - the presence of many identical genes permits the corresponding transcripts to be made a high rate?
Xenopus has about 900 rRNA genes in each cell.
What are 2 examples of PoC 5: Gene amplification?
1.) rRNA genes in the Xenopus oocyte are multiplied 1000-fold.
2.) Tumor cells can sometimes become resistant to Methotrexate by amplification of DHFR genes.
What are 3 examples of PoC 6: Specific genes can be activated for transcription?
1.) Different tissues contain differing quantities of mRNAs.
2.) Sometimes due to transcription factors (Ex. Steroid hormones exert their effects by interacting with TFs.)
3.) Sometimes due to aceylation of histones (Acetylation of histones determines how open/closed DNA, thus regulating access to information.)
What is an example of PoC 7: Primary transcripts may require processing (Alternative Splicing) to produce the active mature mRNA (Ex. Membrane-bound vs. secreted immunoglobulins)?
Different tissues contain differing quantities of mRNAs.
What are 2 examples of PoC 8: mRNA must be transported from the nucleus to the cytoplasm?
1.) Different tissues contain differing quantities of mRNAs.
2.) Cancer cells take advantage of this. There is no RNA restricted to nucleus in cancer so end up with uncontrolled translation into proteins.
What are 2 examples of PoC 9: Rate of mRNA degradation?
1.) Hemoglobin mRNA has a half-life of 100 days while the HeLa cell mRNA has a half-life of several hours.
2.) Iron regulates the half-life of human transferrin receptor mRNA.
What are 2 examples of PoC 10: mRNA in the cytoplasm may be masked in an inactive form?
1.) Maternal mRNA in eggs remains inactive until after fertilization.
2.) Iron regulates whether or not human ferritin mRNA is translated.
What are 2 examples of PoC 11: Rate of translation?
1.) The alpha and beta chains of Hemoglobin are translated at different rates.
2.) Heme regulates initiation of translation.
What is an example of PoC 12: The post-translational fate of a protein may depend on the class of ribosomes on which it is synthesized?
Proteins to be secreted or incorporated into the membrane are synthesized on ribosomes bound to the rough endoplasmic reticulum.
What is an example of PoC 13: Protein modification?
Proinsulin is cleaved to form active Insulin.
What are 2 examples of PoC 14: Protein degradation rate?
IgG antibody half-life = 25 days.
IgE antibody half-life = 25 days.
What are 3 examples of regulation of eukaryotic gene expression by chromatin modification?
1.) Histone acetylases favor gene expression and deacetylases favor inactive chromatin.
2.) Scaffolding proteins that condense regions of chromatin favor inactive chromatin.
3.) DNA methylating enzymes favor inactive chromatin.
How does DNA methylation help turn off genes?
The binding of gene-regulatory proteins and GTFs near an active promoter prevents DNA methylation by some unknown mechanism. If most of these sequence-specific DNA-binding proteins dissociate (as generally occurs when gene is turned off), DNA becomes methylated, enabling other proteins to bind and shut down the gene completely.
What is the most common type of gene regulation in eukaryotes?
Regulation of the initiation of mRNA transcription is the most common type of gene regulation in eukaryotes.
What is the mechanism of action for regulating the initiation of mRNA transcription in eukaryotes?
Like prokaryotes, the rate of mRNA transcription is regulated by the interaction of regulatory proteins that bind DNA near the promoter and modulate the rate at which the transcription initiation complex can form on the promotor.
What are the DNA sequences to which activator proteins bind in eukaryotic DNA?
Activator sequences bind response elements. A few response elements are located within the promoter region (UPE) but most are clustered outside the promoter to form an enhancer region that allows control of gene expression by multiple signals.
What are Upstream Promoter Elements (UPEs)?
Eukaryotic cells have UPEs that bind transcriptional regulator proteins (similar to CAP protein in prokaryotes). Only the proximity of the UPE to the -25 TATA box sequence distinguishes it from an enhancer.
What do UPEs include?
1.) A CCAAT box (around -75) that binds transcription factor NF-1.
2.) A GC-rich sequence that binds a general transcription factor SP-1.
What is an Enhancer?
An Enhancer is a sequence of bases in DNA that is a collection of binding sites (modules) for binding gene-regulatory proteins.
What are Response Elements in an Enhancer?
What is an example of a Response Element?
1.) Response Elements are the bases sequences that serve as binding sites for gene-regulatory proteins.
2.) A gene that is activated in response to a steroid hormone will have a Hormone Response Element (HRE).
What is the difference between an Upstream Promoter Element and an Enhancer?
The difference between a UPE and an Enhancer is location in relation to the -25 TATA box.
The UPE has a CCAAT box around -75.
The Enhancer may be up to 1000 base pairs from the gene of interest.
Where is an Enhancer located in respect to its gene?
An Enhancer stimulates transcription of the gene under its control and may be located upstream, downstream, or within the intron (immunoglobulin genes) but the orientation of the Enhancer in respect to the gene does not matter.
What are 2 other features of Enhancers?
1.) Enhancers can act in a tissue-specific manner if the DNA-binding protein that interacts with them is only present in certain tissues.
2.) Enhancers can be targets for steroid hormones. (Glucocorticosteroids work by binding intracellular receptors and this complex binds an Enhancer.)
How are Enhancers brought close to the basal promoter region?
Bending of the DNA molecule brings Enhancers close to the basal promoter region.
What are Silencers?
Silencers are sequences similar to Enhancers that bind repressor proteins in eukaryotes.
What are Transcription Factors?
Transcription Factors are proteins that bind to regulatory DNA sequences and to other Transcription Factors or to RNA polymerase 2 in order to regulate initiation of transcription.
What are General Transcription Factors?
In eukaryotes, General Transcription Factors (GTFs) must bind to the promoter to allow RNA polymerase 2 to bind and form the initiation complex at the start site for transcription. (The GTF TFIID must bind the TATA box before RNA polymerase 2 can bind.)
What are Specific Transcription Factors?
Specific Transcription Factors bind to regulatory DNA sequences (Enhancers, Silencers) and modulate the formation of the initiation complex, thus regulating the rate of initiation of transcription.
What determines which genes will be transcribed and at what rates?
Each gene contains a variety of Enhancer/Silencer sequences in its regulatory region. The exact combination of Specific Transcription Factors available and active in a particular cell at a particular time determines which genes will be transcribed and at what rate.
What can also regulate the activity of Specific Transcription Factors.
Hormones can also regulate the activity of Specific Transcription Factors.
Transcription Factors contain what 2 recognizable domains?
1.) DNA-binding domain
2.) Activation domain
To what does the TF DNA-binding domain bind?
The DNA-binding domain binds to a specific sequence in the promoter, enhancer, or silencer.
What are the 5 common TF DNA-binding domains?
1.) Zinc fingers (seen on steroid hormone receptors)
2.) Leucine zippers
5.) Homeodomain (encoded by Homeobox (HOX) or Homeotic genes and seen on some Transcription Factors involved in development.)
Why does an Attenuator Stem between regions 3 and 4 signal transcription to stop?
The regions between 3 and 4 are GC rich.
What is the function of the TF activation domain?
The activation domain allows the Transcription Factor to bind to another Transcription factor or to RNA polymerase 2 to stabilize the formation of the initiation complex.
What are the Cis Regulators?
The DNA regulatory base sequences (promoters, enhancers, response elements, and UPEs) in the vicinity of a gene that serve as binding sites for proteins are the Cis Regulators.
What are Trans Regulators?
What makes them special?
1.) Transcription Factors and the genes that code for them are called Trans Regulators.
2.) Trans regulatory proteins can diffuse through the cell to their point of action.
What is the general composition of Enhancers, UPEs, and Transcription Factors (Figure on page 19)?
What makes up the Promoter Region?
Where do General Transcription Factors bind?
Where do Specific Transcription Factors bind?
1.)From right to left, Transcribed Region is +1, TATA Box is -25, UPE is next, CAAT Box is -75. Enhancer can be downstream, upstream, or in intron as long as it is on the same DNA.
2.) The TATA Box, UPE, and CAAT Box make up the Promoter Region.
3.) General TFs bind the TATA Box.
4.) Specific TFs bind the UPE, CAAT Box and Enhancer.
What is special about TFIID?
TFIID is a General Transcription Factor that must bind the TATA box before RNA pol 2 can bind the Initiator.
(It is also called the TATA Factor.)
What is the mechanism of action for
TFIID and RNA pol 2?
1.) TFIID binds TATA Box.
2.) RNA pol 2 binds Initator.
3.) Specific Transcription Factors bind Enhancer.
4.) DNA folds back on itself to bring the Enhancer close to the Promoter Region.
5.) Transcription begins.
What is important to remember about Enhancers and their Specific Transcription Factors?
Enhancers can be downstream, upstream, or within the intron. Specific TFs interact in different ways to turn genes on or off.
How long is a typical DNA-binding Site?
A typical DNA-binding Site is usually 6 to 10 base pairs in length.
How is the TF Myc processed from the inactive form to the active form?
Myc is activated via protein synthesis, so there really is no inactive form. It is generated when needed.
How is the TF Steroid Hormone Receptor processed from the inactive form to the active form?
Steroid Hormone Receptor is activated via ligand binding.
How is the TF jun processed from the inactive form to the active form?
jun is activated via protein phosphorylation.
How are the TFs fos and jun processed together from their inactive forms to their active forms?
fos and jun are activated via the addition of a second subunit. (fos binds the DNA, then jun binds to activate fos.)
What is the mechanism of action for the Steroid Hormone Receptor?
Steroid Hormone Receptors are inactive TFs in the cell. Steroid Hormone will enter the cell and bind the Steroid Hormone Receptor to activate it and form a complex. The activated complex is transported to the nucleus where it binds Hormone Response Elements on the Promoter and initiates transcription.
What are Zinc Fingers?
Zinc Fingers are TFs in which Zinc makes a loop among cysteines.
What is important to remember about Zinc Fingers?
Steroid Hormone Receptor is a Zinc Finger!
What is important to remember about Helix-Turn-Helix and Leucine Zippers?
Helix-Turn-Helix and Leucine Zippers can can only be part of a TF due to their composition.
What four patterns of Alternative Splicing have been observed to produced distinct mRNA?
What is the mechanism of action?
1.) Optional exon, optional intron, mutually exclusive exons, and internal splice site patterns have been observed.
2.) In each, a single type of RNA transcript is spliced in two alternative ways to produce two distinct mRNAs.
(See page 23 in Notes)
How is Alternative Splicing involved in antibody synthesis?
The location of RNA cleavage determines whether or not the transcript will be long or short.
If the transcript is long, the intron is removed and the terminal peptide will be hyrdophobic, thus generating a membrane bound antibody. (Unstimulated)
If the transcript is short, the intron is not removed (acceptor splice junction is missing) and the terminal peptide is hydrophilic, thus generating a secreted antibody. (Stimulated)
What is the best example of eukaryotic regulation of translation?
The best example is the regulation of synthesis of human ferritin and human transferrin receptor via iron concentration.
What is the relationship between the presence of Iron and the presence of Ferritin (binds Iron) or Transferrin Receptor (imports Iron)?
If low Iron - Ferritin down, Transferrin Receptor up
If high Iron - Ferritin up, Transferrin Receptor down
What makes up the Iron Response Elements (IREs) on Ferritin and Transferrin?
What happens when regulatory proteins bind them?
1.) Ferritin's IRE is made of CGU and is located in the 5' untranslated region. Protein binding here blocks translation of Ferritin mRNA.
2.) Transferrin's IRE is made of C and is located in the 3' untranslated region. Protein binding here blocks degradation of Transferrin mRNA.
What happens in Iron Starvation (low Iron)?
TF binds IRE in 5' region of Ferritin mRNA and blocks tranlastion of Ferritin mRNA so no Ferritin is made.
The same TF binds IRE in 3' region of Transferrin mRNA, stabilizing and protecting the ends from degradation to initiate translation of Transferrin mRNA to make Transferrin Receptor.
What happens in Excess Iron (high Iron)?
Iron binds the TF in the 5' region of Ferritin mRNA and causes a conformational change that dissociates the TF from the IRE so that Ferritin mRNA is translated and Ferritin is made.
Iron binds the same TF in the 3' region of Transferrin mRNA and a causes a conformation change that dissociates the TF from the IRE so that Transferrin mRNA is no longer stable or protected from degradation and no Transferrin Receptor is made.
What is RNA Interference (RNAi)?
What are 2 types of RNAi?
1.) RNAi is achieved by small RNA molecules that bind to the 3' untranslated region of an mRNA molecule to interfere with Translation by making a double strand.
2.) MicroRNA (miRNA) and ShortInterfering RNA (siRNA)
What is miRNA?
miRNA is a new discovery of RNAi that is 19-25 nt long, endogenous in cells, can be an imperfect match to region of interest, and inhibits translation of either several genes at once or a single specific gene. miRNA is implicated in cancer and other diseases. (Antioncogene P53 slows down cell cycle progression but miRNA can interfere with Antioncogene P53 and perpetuate cancer.)
What is siRNA?
siRNA is a form of RNAi that is made in a lab, is only 19-21 nt long, must be an exact match to its region of interest, and leads to mRNA degradation. siRNA is used in the lab to prevent synthesis of individual proteins and is a potential therapy for treating cancer and viruses.
How are Epigenetics a form of gene regulation?
What are the most common forms?
1.) Epigenetics are changes in gene expression or cell phenotype that are not sequence based but can be inherited.
2.) The most common forms are DNA methylation and Histone Modification.
How is DNA methylation a form of Epigenetics?
Methyl marks added to certain DNA bases repress gene activity.
(DNA is packaged in nucleosomes. Cytosine methylation to make chromatin more condensed can then be passed to progeny.)
How are Histone Modifications a form of Epigenetics?
What is Histone Code?
1.) A combination of different molecules can attach to the tails (Histones) of proteins and alter the activity of the DNA wrapped around them.
2.) Histone Code: Relaxes DNA and turns genes on.
What are two other examples of Epigenetics?
1.) X-Chromosome Inactivation results in Barr Bodies because on the X chromosomes in each cell of a female is turned off.
2.) Genomic Imprinting is a natural phenomenon in which certain genes are only expressed when inherited from one of the two sexes.
How is Genomic Imprinting partly responsible for Prader-Willi and Angelman Syndromes?
Some genes are transcriptionally active only when transmitted by one of the sexes. The gene from the other parent is inactivated by methylation during gametogenesis. When a deletion occurs in a chromosome with the single active copy, there are no active copies of the gene and the disease occurs.
What is Prader-Willi Syndrome?
Prader-Willi Syndrome is a 4 megabase deletion on Chromosome 15q that is transmitted by the father (deleted gene) and presents with moderate mental retardation, hypogonadism, small hands/feet, obesity.
(See page 28 in Notes for inheritance pattern!)
What is Angelman Syndrome?
What else is it called?
1.) Angelman Sydrome is a 4 megabase deletion on Chromosome 15q that is transmitted by the mother (deleted gene) and presents with severe mental retardation, seizures, ataxic gait, behavior disorders.
2.) It is also called Happy Puppet Syndrome.
(See page 28 in Notes for inheritance pattern!)
What is the schematic of the Prader-Willi and Angelman inheritance pattern?
Square = Male, Circle = Female
Small rectangle = PW, Small oval = Angel
Open = Active, Filled = Imprinted, Missing = Deleted
What is the inheritance pattern of a healthy son in regards to Prader-Willi and Angelman Syndromes?
A is normally imprinted in father. PW is normally imprinted in mother.
A healthy son receives active A from mother and active PW from father. (See page 28 in Notes)
What is the inheritance pattern of a Prader-Willi son?
A is normally imprinted in father. PW is normally imprinted in mother.
A Prader-Willi son receives active A from the mother but active PW is deleted from the father so Prader-Willi Syndrome presents. (See page 28 in Notes)