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134 Cards in this Set
- Front
- Back
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Best known prokaryotic genome
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E. coli genome
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How many base pairs are in the genome?
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4,639,221
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1Mb
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1,000,000 bp
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1 Kb
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1,000 bp
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1 bp
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2 nucleotides
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What does oriC refer to?
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Origin of replication; found opposite of TER
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What does TER refer to?
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Replication termination; found opposite of oriC
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Why is an understanding of the prokaryotic genome important?
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1. Bacteria occupy all environments and have the ability to respond rapidly to changing environments
2. An understanding of genes expressed in pahogenic bacteria may allow drugs to be developed to alter the expression of these genes 3. Model organism--used for scientific study |
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Why is it not healthy for all of the genes in a cell to be expressed at once?
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If all genes were expressed at once in a cell it would waste energy, inability to react to enviornment and it's a poor use of resources.
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What are the ways gene expression is controlled?
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1. Transciptional control
2. Translational control 3. Post-transational control 4. Some genes are continually expressed (constitutively) |
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Transcriptional control
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No mRNA is made
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Translational control
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mRNA is made but it does not get translated
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Post-translational control
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Protein is made but is not activated by chemical modifications
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Constitutively
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Enzmes used in glycolysis
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Regulatory protein
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A protein that directly bonds to the DNA to enhance or terminate transcription
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Repressor
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The proteins that inhibit transcription
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Negative control
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When regulation is controlled by a repressor
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Activator
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The proteins that increase the rate of transcription
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Positive control
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When regulation is controlled by an activator
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Effector molecule
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Molecules that do not bond directly to the DNA but bind to the repressor or activator and affect their ability to bond to DNA
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Inducer
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Small molecule that increases transcription by binding to a repressor protein to stop its repression OR binds to an activator to allow it to bond to the DNA to increase transcription
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Inducible gene
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Genes controlled by an inducer (turned off unless needed)
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Corepressor
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Small molecule that bonds to a repressor and enhances its ability to bond to DNA and repress transcription
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Inhibitor
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Small molecule that binds to an activator and does not allow it to bond to DNA and allow transcription
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Repressible gene
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Genes controlled by co-repressors or inhibitors (turned on unless not needed)
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What is the relationship between E. coli and lactose?
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E. coli needs a sugar source to perform respiration (production of ATP). Prefer glucose but not always available. E. coli could metabolize lactose as a sugar source
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Who discovered the mechanisms behind lactose metabolism?
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Jacques Monod and Francois Jacob
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What is B-galactosidase and how is it produced?
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B-galactosidase is induced in presence of lactose
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What does a master plate contain?
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Nutrient agar
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What can grow on a master plate?
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Anything
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What does a replica plate contain?
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Minimal media
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What can grow on a replica plate?
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Only wild type; no mutants
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How are bacteria transferred from one plate to another?
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Press master plate on velveteen on wooden block (attached with plastic hoop). Minimal plate is pressed on block. Pressing velveteen makes a photocopy
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Lac Z wildtype function
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Codes for B-galactosidase which breaks down lactose
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Lac Z mutant function
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Does not produce B-galactosidase
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Lac Y wildtype function
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Codes for galactosidase/lactose permease
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Lactose permease
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Protein that embeds itself in cell membrane and transports lactose into cell
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Lac Y mutant function
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Does not produce permease to allow lactose into cell
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Lac A wildtype function
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Codes for transacetylase - function unknown
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Lac A mutant function
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Unknown
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Lac I wildtype function
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Produces protein that stops lac Z and lac Y genes from being used. Codes for repressor
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Lac I mutant function
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Does not produce protein and lac genes not regulated
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Lac O wildtype function
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Location where protein from lac I bonds to stop lac Z and lac Y
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Lac O mutant function
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Lac I protein does not recognize proper location and lac genes are not regulated
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Operator
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A specific DNA sequence that the repressor recognizes to bond with the DNA and stop transcription
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What type of molecule is lactose and what does this make the lac genes?
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When lactose is added, it bond to the repressor or protein and changes its shape. This makes lactose an inducer.
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What occurs to the operator and repressor in the presence of lactose?
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The repressor protein can no longer bond to the operator to stop RNA polymerase. The lacZ, Y, and A genes can now be transcribed by the RNA polymerase
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What is an operon and what makes up an operon?
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Refers to a set of two or more genes that are controlled by a single promoter and are transcribed together into one mRNA strand. An operon contains a promoter, operator, structural genes, and a terminator
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trpE, D, C, B, and A
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All code for the enzymes used to make tryptophan
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trpR
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Codes for the repressor (but not functional)
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trpO
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DNA sequence where the repressor binds to the operator
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trpL
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Involved in a process called attenuation
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What is attenuation?
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Terminates the mRNA before the entire operon is transcribed (another way to control the operon)
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What type of molecule is tryptophan and what does this make the trp genes?
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In the trp operon, tryptophan acts as a corepressor and will bond with the repressor
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What occurs to the trp operon when tryptophan is present?
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When tryptophan bonds with the repressor, the repressor takes the correct shape to bond with the DNA and stop the operon
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Why is the trp operon useful?
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This operon allows the bacteria cell to make tryptophan in a tryptophan-poor enviornment but stops synthesis in a tryptophan-rich environment
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What is a translational repressor?
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A protein that bonds to the mRNA and stops translation
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What are two ways translational repressors hinder translation
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1. Binds close to the Shine-Delgarno site or start codon and blocks the ribosome's ability to bond to the mRNA
2. Binds outside the Shine-Delgarno site or start codon but causes mRNA to have a secondary structure which would not allow it to bond to the ribosome |
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What is antisense RNA?
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RNA that is complementary to a strand of mRNA. Does not code for a protein--it only bonds to a functional piece of mRNA to inhibit translation
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How does osmoregulation in bacteria use antisense RNA as a translational control?
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Osmoregulation in bacteria controls the amount of H2O in the cell. ompF gene codes for a protein involved in osmoregulation. micF is a gene that codes for a piece of mRNA that matched with ompF's mRNA. When the amount of the protein produce in ompF needs to be low, the transcription of micF is high. micF is complementary to the ompF mRNA and the two form hydrogen bonds. This bonding does not allow the ompF mRNA to bond to the ribosome and translation does not occur
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How many structural genes are in the trp operon
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5 - trpE, trpD, trpC, trpB, trpA
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What type of protein can bind to the operator?
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Repressor
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What occurs when tryptophan is lacking in the enviornment?
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The repressor is inactive RNA polymerase binds to the promoter site and then proceeds down the DNA, transcribing the genes for the tryptophan bio synthesis enzymes
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What does tryptophan bond with to control the amount of tryptophan in the cell?
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Repressor
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How does the active repressor stop transcription?
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The active repressor binds to the operator, located within the tryptophan promoter and blocks transcription
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Why can an active repressor bind to the DNA vs. an inactive repressor and its inability to bind to DNA
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When tryptophan is absent from the environment, the repressor is in an inactive conformation and cannot bind to the DNA to prevent transcription
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When is the lac operon used and why is it only used at this time?
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The lac operon is used when lactose is available to be metabolized. It's only used then so it does not waste energy
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When is the lacI gene expressed?
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In the absense or presence of lactose
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What occurs in the absense of lactose?
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The lac repressor binds to the lac operator site
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How does the repressor and operator work?
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Repressor binding to the operator blocks progression of RNA polymerase, like a DNA road block
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Why are the corresponding proteins made?
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RNA polymerase is unable to transcribe the lac structural genes?
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What occurs when lactose is present in the cell?
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It binds to the allosteric site of the lac repressor. This changes the conformation of the repressor
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Why can RNA polymerase transcribe when lactose is present?
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The repressor can no longer bind to the lac operator site; RNA polymerase is able to transcribe structural genes
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What is coded by the Z and Y genes?
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Proteins required for the metabolism of lactose
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What must occur in order for the bacteria to use lactose?
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The bacteria must first alter its metabolism. The bacterium must turn on several genes, found in the lac operon, which are required for lactose metabolism
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What is the inducer in the lac operon?
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Lactose
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How is the trp operon different from the lac operon?
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The trp operon expresses genes in the operon until a repressor becomes activated and turns the expression off
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What does attenuation prevent and when is this prevention needed?
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Attenuation in the trp operon can prevent expression of tryptophan biosynthesis genes when tryptophan level in the cell are high
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What trp gene is involved in attenuation
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trpL
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What do interactions between complementary mRNA sequences create? How many regions are involved?
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Stem-loop structures in the RNA transcript. Four regions are involved.
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What is region 2 complementary to? What is the restriction?
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Both regions 1 and 3. Region 2 can form a stem-loop structure with either of them but not with both at the same time
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What is region 3 complementary to?
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2 and 4
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What occurs when regions 1 and 2 bond together?
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Region 3 forms a stem-loop with region 3. Immediately following region 4 is the attenuator
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What is the attenuator?
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A uracil-rich sequence
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What does the interaction between the 3-4 stem loop and attenuator cause?
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When region 3-4 stem loop is present, it acts with the attenuator to form a terminator for transctipion at the end of the trpL gene
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What occurs when the 2-3 stem loop is present?
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When the 2-3 stem loop is present, the terminator structure does not form and transcription proceeds to the trpE gene and other tryptophan biosynthesis genes in the operon
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When transcription occurs alone, what form does the trpL mRNA take? How many loops are present and what occurs?
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If transcription is occuring alone (in the absense of translation), then the trpL mRNA will fake on its most stable conformation; the region 1-2 and 3-4 stem-loops form and transcription is terminated at the end of the trpL gene
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What determines stalling?
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Two adjacent tryptophan codons in region 1 determine whether or not stalling will take place there
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What occurs if tryptophan levels are low? How does this affect the ribosome?
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Tryptophan transfer RNA levels will also be low. This will cause the ribosome to pause at the two tryptophan codons as it waits for tryptophan transfer RNA
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What loop will form when the ribosome pauses? What does this then allow the cell to do?
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The location of the ribosome at this pause will shield region 1 from region 2, thus freeing region 2 to form a stem-loop with region 3. Since this prevents the formation of the tryptophan biosynthesis genes in the operon
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What happens if tryptophan levels are high?
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The ribosome will not get stalled at the tryptophan codons in region 1. The ribosome will continue until it reaches the stop codon for the trpL gene, where it will experience the normal pause that occurs during translation termination. The ribosome will block region 2 from region 3. Region 3-4 stem look will readily form
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4 ways to regulate genes in eukaryotes?
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Regulate the process of transcription; regulate the mRNA after it is transcribed; regulate the process of translation; regulate protein stability
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Sub categories of regulating the process of transcription
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Transcription factors, change chromatin structure, DNA methylation
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Sub categories of regulating the mRNA after it is transcribed
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Alternative splicing, mRNA stability, RNA interference (RNAi)
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What are transcription factors?
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Broad term used to describe proteins that influence the ability of RNA polymerase to transcribe a gene
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General transcription factors
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Proteins required for the binding of RNA polymerase to the promoter region ("docking" station for RNA polymerase) for any gene--they are required no matter the type of cell or gene
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General transcription factor example
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TFIID - binds to the TATA box and causes a shape to change in TFIID and a shape change in DNA, which then allows other transcription factors to bind
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Regulatory transcription factors
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Additional proteins taht can bind to a different sequence of DNA (not the TATA box) near teh promoter region
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Activator
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Can enhance transcription - binds to a DNA sequence called the enhancer
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Enhancer
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Piece of DNA that activator bonds to
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Repressor
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Can prevent transcription and binds to a DNA sequence called the silencer
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Silencer
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Piece of DNA that repressor binds to
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What is an example of coordinated expression of genes?
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Enhancer and silencer sequences help cells coordinate the expression of groups of genes that are far apart or on separate chromosomes. The coordination of genes using the same transcriptoinal factors can be found in plants when they respond to drought. Synthesizes a number of proteins at once and theya re scattered throughout the genome - dehydration response element
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What are epigenetics?
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Genes that are regulated by reversible, non-sequence DNA alterations
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What are 3 examples of epigenetics?
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Chromatin compaction, DNA methylation, and DNA interference (RNAi)
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Histones
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Like spool of thread - a positively charged protein that the DNA is wrapped around
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Nucleosome
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The assembly of histone proteins and DNA
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Open conformation
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Euchromatin - the accessible DNA for the cell to use and transcribe
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Closed conformation
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Heterochromatin
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Describe the structure of a histone
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Has a "tail" of ~20 amino acids that sticks out of the structure and contains positively charged amino acids
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How does histone acetyltransferase and histone deacetylase affect histone structure?
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Enzymes, known as histone acetyltransferases, can add acetyl groups (CH2CO) to the tails. The acetyl groups neutralize the positive charge of the tails, chainging the structure of the histone and loosening the DNA around the histone allowing it to be transcribed. The acetyl groups can be removed with another enzyme (histone deacetylase) and the DNA will be in closed form
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What other functional groups can affect the structure of histones?
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Histone structures can also be changed through methylation (CH3) and phosphorolation (PO4)
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What is the function of NDA methyltransferase?
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Methyl groups are added to bases, mainly cytosines. The enzyme responsible for adding methyl groups is DNA methyltranferase, which adds a methyl group to cytosine
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What is the difference between hemimethylation and full methylation?
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If only one strand is methylated, it is called hemimethylation. If both stransd are methylated it is full methylation
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What is a CpG island?
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A dinucleotide of C and G in DNA that are connected by phosphodiester bonds. Contain 1000-2000 bps and contain a LOT of cytosine and guanine dinucleotides
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How do CpG islands relate to DNA methylation (what silences a gene vs. activates it)?
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Evidence suggests that methylated CpG islands will silence genes whle the unmethylated islands have active genes; housekeeping genes and tissue-specific genes
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Housekeeping genes
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Gnes that encode proteins required in most cells and the CpG islands are unmethylated by their promoters
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Tissue-specific genes
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-
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What are the two ways that DNA methylation can silence a gene?
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Prevents the binding of transcription factors to the promoter region and changes DNA from an open conformation to a closed conformation
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Prevents the binding of transcription factors to the promoter regoin
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Methylated CpG islands could prevent the binding of an activator to an enhancer sequence and this would not allow the RNA polymerase to transcribe a gene
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Changes DNA from an open conformation to a closed conformation
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Proteins, known as methyl-CpG-binding proteins, bind to the methylated sequence. Once bound, the proteins recruit other proteins (histone deacetylase) that causes chromatin to become compact
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What is the function of maintenance methylase?
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Catalyzes the formation of methylcytosine (methylated cytosine) on the new DNA strand when DNA is replicated for cell division
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What is an example of maintenance methylation (mention Barr Body)?
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Maintenance methylation is seen in human female cells in which one of the x chromosomes are randomly inactivated (known as Barr body) and becomes heterochromatin. If one of the x chromosomes is NOT methylated, there will be too much protein product from 2 x chromosomes
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De novo methylation
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Methylation of previously unmethylated DNA
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Demethylase
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Causes DNA methylation (very rare)
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Why is it a good thing that de novo methylation and demethylase are a rarity?
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This rarity preservse what genes are turned on and what are turned off from generation to generation. This ensures that cells in specific tissues continue with the correct function and do not start making unneeded proteins
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What is alternative splicing and why is this advantageous?
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RNA can be spliced in more than one way. Alternative splicing allows 2 or more proteins to be made from one mRNA strand. Possible evolutionary advantage - allows an organism to carry fewer genes in its genome. Possible link to organism complexity. Chimps and humans have the same sized genome but humans have more alternative splicing
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How does the stability of mRNA affect the protein product it codes for?
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Life-span can be altered by altering the mRNAs stability and better stability means that the mRNA will produce more protein product
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How does the polyA tail affect mRNA stability
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PolyA-binding protein recognizes the polyA tail on mRNA and bonds to the polyA tail enhancing its ability ***
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What is microNRA (miRNA)
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RNA molecules that do not direct the synthesis of a protein. Vary in size from 21-22 nucleotides in length. First found in c. elegans but have now been found in many eukaryotes such as plants and humans.
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What has microRNA been linked to?
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Have been linked to silencing coding mRNA by binding with the coding mRNA and it can no longer be translated
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What are the stops to how miRNA silences genes?
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1. DNA is transcribed into RNA, which folds over itself forming a hairpin
2. The enzyme dicer cuts the dsmRNA into smaller pieces 3. Half of the dsRNA is degraded and hte other half associates with a protein called RISC, which allows the miRNA to find its complementary half 4. The RISC and miRNA bonds to any mRNA with the complementary sequence to the miRNA bonded to the RISC 5. The translation of the mRNA is blocked OR the RISC complex degrades the mRNA |
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RNA interference
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The blocking of mRNA by miRNA
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What are 3 possible advantages to having RNAi in a cell?
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1. Another way to regulate genes: When the gene for the miRNA is turned on, it will silence another gene
2. Defense mechanism against viruses: Some viruses inject RNA into its host cell and the miRNA could bond ot this RNA and stop the infection 3. Silencing transposable element: transposable elements are segments of DNA that just randomly insert themselves into the genome and RNAi could silence the RNA product of these elements |
