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lac operon ecoli experiments
1). what is the purpose of adding xgal in the medium ?
2). blue indicates lac operon ____
3). no change indicates lac operon ____
4). what is the purpose of treating ecoli with a mutagen?
1). substrate for bgal that when transcribed turns a blue color, allows you an easy indicator if lac operon is being transcribed
2). is functioning
3). is not expressing enzymes
4). mutate the lac operone so it becomes dysregulated
1). what would you primarily see in a petri dish with no lactose, + xgal + mutagen?
2). what would you primarily see in a petri dish with lactose, + xgal + mutagen?
1). primarily would see no expression (white_ because mutagen doesnt mutate every bacterial cell) but will see some blue colonies because lac operon is unable to be repressed
2). primarily would see expression (blue because mutagen doesnt mutate every bacterial cell) but will see some white colonies because lac operon was inhibited by mutations
Lac operon mutations
1). lacI- mutants have ______ of bgal/permease. in the presence and/or absence of lactose? why?
2). lacOc mutants have ______ of bgal/permease. why?
3). LacIs mutants have ______ of bgal/permease. in the presence and/or absence of lactose? why?
1). constitutive expression due to inability of repressor to bind the operator to regulate the operon in BOTH presence and absence of inducer
2). constitutive, operator is mutated and unable to bind the normal repressor, in presence and absence of both
3). repression in presence and absence because it is unable to bind allolactose (inducer), so it is constantly bound to the operator and blocks transcription
Trans vs cis
1). what are trans-acting elements? are they stationary (in one part of the cell) or do they travel? do these alter one vs multiple strands gene expression?
2). what are cis-acting elements? are they stationary (in one part of the cell) or do they travel? do these alter one vs multiple strands gene expression?
3). what is an example of trans and cis acting elemenets?
1). can travel through cytoplasm and into nucleus to act at target DNA sites. can bbind to many DNA sequences on different strands and chromosomes and alter gene expression
2). parts of a DNA sequence that when bound to trans acting elements can influence the expression of adjacent genes on the SAME DNA molecule
3). trans-acting = proteins, like transcription factors/ transcription activators/transcription repressors (LacI); bind to stationary molecules like DNA (like the operator!!
Ecoli partial diploid
1). what do partial diploid ecoli lines exploit?
2). if the operator is mutated of o ne of the cell lines, does this affect expression of one or both lines?
1). inserting a plasmid with the trans activity of the repressor (laci and mutants) can translocate to alter the expression of original lac operon
2). only affects expression of the genes attached to that operator
lac repressor binds operator DNA
1). when they radioactively labeled the repressor and introduced a WT lac operon, where did they see radioactivity in the test tube?
2). when they radioactively labeled the repressor and introduced a lac Oc mutated lac operon, where did they see radioactivity in the test tube?
3). how can DNAse 1 footprinting be used to identify the area of the repressor binding region (operator)? what does the repressor do?
1). DNA and radioactivity colocalized, dna band is radioactive
2). dna does not bind repressor due to mutation, no radioactiviy present in DNA band
3). use multiple restriction sites throughout the lac operon with DNAse 1 treatment enzymes without repressor bound to create a map. Then, add the repressor and do the DNAse treatments. the repressor binds the DNA and protects it from enzymatic digestion, so there is a region of the DNA ladder that is lacking bands.
Helix turn helix motif
1). what is notable about thees HTH motifs that is required for laci binding to laco?
2). describe this motif and where it binds DNA? what part of the reg protein binds?
1). specific AA sequence to bind to laco
2). two alpha helicies seperated by a turn, fits in the major groove of DNA and only one alpha helix recognizes specific DNA sequences
Lac Repressor and Operator
1). how many subunits of lac repressor is needed to bind to the operator? how many HTH motifs are there?
2). how many operators does the lac operon have? how many repressors does each bind
3). how does the above alter repression of genome?
1). tetramer = four subunits = 4 HTH motifs
2). three operators. O1 = strongest aff and binds 2 subunits, O2 *
* O3 (lo ated upstream) bind the other two subunits
3). maximal repression occurs when all 4 subunits are bound = forms a loop in the DNA and prevents rna poly from binding
Lac operon is also regulated by glucose
1). what is the expected effect of glucose presence on lac operon expression?
2). what is the function of cAMP receptors beign able to bind the promoter?
3). in the absence of glucose cAMP is ____________. what does this do to the ability of the lac operon to be expressed?
4). in the presence of glucose cAMP is ____________, why? what does this do to the ability of the lac operon to be expressed?
1). repression, have glucose to utilize so why make eztra enzymes!
2). when bound, camp Receptors bind and recruit RNA polymerase to the operon
3). high = binds receptors = two dimerize to form complex -> DNA = promotes lac operon expression
4). low because glucose depletes adenylyl cyclase = reduces camp = no binding of receptor = no recruitment of rna poly = repression of lac operon
1). why does the repressor bound to the operator prevent RNA polymerase from binding?
2). why does CRP-camp complex promote rna poly binding?
3). can RNA poly bind in the absence of CRP-camp complex bound to DNA?
1). rna poly binding site and operator site overlap, so when repressor is bound RNA poly cant bind
2). upstream of rna poly binding site = allows rna poly to recognize and facilitates binding
3). yes, but it is less likely to unwind DNA and initiate transcription
Lac operon fusions
1). how can the lac operon be exploited to regulate expression of other genes?
2). what is the common way for this new DNA to be introduced to ecoli?
3). what is the purpose of IPTG instead of using lactose?
1). make recombinant dna with structural genes you ned to express attached to the lac operon, and then control expression in cell
2). utilization of plasmid vector
3). structurally similar but doesnt get degraded = allows for more consistant production of gene of interest
Lac z as a reporter gene
1). why is lac z an important reporter gene?
2). why is lac z commonnly fused with other operators when identifying gene regulation?
1). easy to assay, can measure gene expression or identify gene regulatory elements
2). can use assays to easily identify if the operon was encoded like the color test.
1). how is this operon regulated by the presence of tryptophan in the medium and why?
2). what are the functional trp genes that synthesize the AA?
3). what is the function of P and O?
4). What is trpL? what is this used for
5). what does trpr encode?
1). operon is turned off if trp is in the medium because the organism doesnt want to synthesize something if it doesnt have to (conserve energy), but this AA is essential for life
3). P = promoter, binds polymerase; o = operator, binds repressor
4). TrpL = trp leader sequence, used in attenuation of trp
5). encodes the repressor protein
Absence of tryptophan
1). what happens to the transcription of trpr?
2). what does the above allow to occur?
3). tryptophan is _______
4). do trpR- mutants result in constitutive expression of tryptophan? why or why not?
1). trp R transcribes but is not an active repressor that cant bind opertaor
2). rna poly can bind promoter = transcribe genes
4). nope! this is because there is a second regulatory element called attenuation
Presence of tryptophan
1). what happens to the transcription of trpr? what does the presence of trp cause?
2). what does the above allow to occur?
3). tryptophan is _______
4). tryptophan in the medium acts as a ______
1). trpR is transcribed, and able to bind 2 trp molecules = conformational change = active
2). this complex is able to bind to the operator = RNA poly cant bind
3). not expressed
4). corepressor! binds to trp repressor = complex that represses
Attenuation of Trp expression
1). When the mRNA becomes truncated during attenuation, where is this terminated at?
2). why is formation of the 1-2 stem loop important for termination of transcription?
3). why does the presence of tryptophan allow the above to occur?
4). when trp is not present, why are 2-3 stem loops allowed to form? what does this do to transcription?
1). leader sequence (trpL)
2) prevents 2-3 from forming = 3-4 stem loop is formed which signals termination
3). presence of charfed trna is available, so when it reaches the trp codon it quickly synthesizes the gene and continues on, which prevents 2/3 from forming
4). when rna poly gets to trp codon on 1 it pauses because no charged trnaTRP to put in there, = allows 2-3 stem loops to base pair = prevents 3-4 stem loop = transcription continues
1). what are these and where are they located?
2). how are these riboswitch activities altered
3). what do they regulate?
1). regulate gene expression = mRNA upstream of initiation codon = aka 5' UTR
2). riboswitch can bind to small molecules via aptamer, and the aptamer state can cause configurations in the expression platform
3). transcription and translation
1). In transcription, what is the sequence that is present when the effector molecule is not bound to the aptamer? what is the fucntion of this?
2). what occurs when the effector molecule does bind the aptamer?
3). what is the purpose of the expression platform?
4). in translation, what sequence must be open on the mRNA for the ribosome to bind?
5). what happens to the above when an effector molecule binds the aptamer?
1). antiterminator, configuration where RNA poly can complete transcription
2). causes antiterminator sequence to switch conformations = now a terminator sequence = termination
3). tells RNA poly whether to continue translating or not
4). the RBS = shine dalgarno sequence in the 5' UTR = translation occurs
5). conform change where RBS is in a stem loop an dunable for ribosome to bind = no translation
1). does this regulate transcription or translation?
2). what do negative regulators do to block translation?
3). what do positive regulators do to promote translation?
2). complimentary BPs to the RBS/shine dalgarno sequence = ribo cant bind, inhibited
3). complimentary BPs with the 5'UTR sequence that nromally keeps the shine dalgarno box occupied and unable to bind to ribos, so now ribosome can bind RBS and translation occurs
Cis acting regulatory elements
1). what is the function of the promoter?
4). how have GFPs been used to demonstrate the effects of this?
1). binds rna polymerase
2). upstrem or downstream that enhances gene expression
3). dna sequences that block the activity of polymerase
4). promoter = basal level of GFP, add enhancer = more GFP,add insulator = less GFP
Trans acting factors- activators
1).what is the function of the activator proteins? what two structural domains must they have?
2). where do these bind?
3). what is their function in basal factors? what facilitates this? what is the function ofh emediator
4). how can coactivators alter chromatin structure?
1). enhance transcription via DNA binding and transcription-activator domains
2). bind to enhancers
3). recruits basal factors (and pol2) facilitated by a protein complex (20+ proteins). The mediator is bound to the enhancer so it can interact with the basal proteins to enhance transcription.
4). binds to activator proteins = displaces nucleosomes = exposes DNA of promoter = gene expression
Trans acting factors- repressors
1).how can these eliminate all basal transcription?
2). how can these supress activity of activator? what is this called?
3). what is quenching?
4). how can these disrupt dimerization of activator?
5). cytoplasmic sequestering?
1). block RNA pol access to promoter
2). compete with activator for same enhancer or will block access of activator to enhance. = indirect repressor
3). activator is bound to enhancer, but binds to the transcription activation domain = cant enhance transcription
4). can bind to activator (heterodimerization) and prevent it from dimerizing which is required for activation of some enhancers
5). keeping it in cytoplasm and unable to bind to nucleus
1). how can these help repress transcription?
2). how do these modify chromatin structurre?
1)prevent RNA pol2 complex (and basal factors) from binding promoter
2). close chromatin and inhibit promoter from being accessed
transcription factor motifs
1). Helix turn helix motif- how does specificity of the TF for DNA determined?
2). how does the helix-loop-helix motif differ?
3). what does each zinc bind in zinc-finger motif?
4). what is notable about the location of leucines on the two alpha helicies? what allows the peptide to be activated? how do these interact with DNA
1). dif AAs interact with certain sequences of DNA = comes from both
2). instead of the turn, it is a loop =longer. , dimers can interact with dna
3). one zn2+ binds on zinc finger beta sheet-helix-beta sheet domain
4). every 7th AA is a leucine = on one side of helix, very hdrophobic = buried on the inside, associated = dimerize at the dimerization domain = zipper formation; then has two DNA binding domains to interact with DNA
Modulation of regulatory TF
3). covalent modifications
1). hormone binding = allosteric effects = can interact with DNA
2). dimers or multimers required for activation
3). post translational modifications, phosphorylation
Eukaryotic regulation of galactose
1). function of Gal7, Gal10, gal1?
2). function of Gal 4? what two binding sites are present?
3). gal 80? what two binding sites are present?
1). structural genes = degrade galactose to glucose to be utilized by the cells
2). gal 4 = transcription activator, has an activation domain, another area for gal80 to bind in inactive state
3). transcription repressor = binds to on transcription activation domain of activator, also binds to gal 1 and gal 3
1). when galactose is absent, what is occuring at the enhancer?
2). what happens to gal 1 and gal 3 when galactose is present?
3). what is allow to occur because of the above and why?
4). when does expression stop?
1). gal 4 cound to gal80 bound at repressor = inactive state
2). allosteric effect = bind to gal 80 = gal 4 activation domain is exposed
3). now RNA poly can bind and genes all controlled by same enhancer are expressed.
4). depletion og galactose = gal 1 and 3 revert back to normal configuration = repression
Steroid Hormone and gene regulation
1). what is unique about steroid receptors?
2). what happens to receptor when ligand binds?
3). what allows the receptor to translocate to the nucleus?
4). where does this bind in the nucleus and what follows? hwo does this regulate transcription of many genes
1). when bound to steroids, they are regulatory transcription factors
2). 2 HSP 90 unbind
3). receptors dimerize = enter nucleus
4). bind to glucocorticoid response elements (all genes have common enhancer sequence)
1). What is a nucleosome composed of? what are these proteins charge?
2). how can chromatin be changed so histones are still present but encouraged DNA expression?
3). how can histones be modified to encourage expression?
1) composed of 8 histone proteins which are basic (+) and bind (-) DNA
2). change in nucleosome position to expose promoter regions
3). either remove a histone octamer or replace with variant histone protein that recruits proteins to encourage DNA expression
1). what does histone acetylation do to expression? deacetylation
2). is HISTONE methylation perfectly indicative of increased or decreased?
3). what does phosphorylation of the histone tail caused?
4). what does ubiquination cause?
1). dNA less tightly wound = increased expression; deacetylation = tightly wound = decreased expression
2). nope! thats DNA (DNA meth = silencing). can be varied
3). loosens up DNA = increased expression
4). targets histone for degradation = removes histones and nucleosomes so DNA is expxosed
1). where does DNA get methylated to be silenced?
2). why are CPG islands usually unmethylated? what does this do to transcription?
3). when do CPG islands become methylated?
1). C5 of CpG islands
2). activators are bound to block access by methyltransferases = RNA pol 2 recruited = transcription activated
3). absence of activators, MeCPs bind to recrruit other proteins to close chromatin = transcription inhibited
1). what does it mean if a gene is paternally imprinted? what gene is expressed?
3). for igf2, if the inslator between IGF2 and enhancer is methylated, is the gene transcribed?
4). if it is methylated? what protein is responsible
1). imprinted = transcriptionally silenced; gene from dad is silenced; maternal is expressed
2). maternally imprinted = moms silenced; paternal is expressed
3). methylated = cant bind CTCF = it is transcribed
4). CTCF binds and blocks enhancer from working = not transcribed
1). where do most miRNAs arise from? how long are they
2). what enzyme acts on pri-miRNA, and what doe this form? where does it go?
3). what enzyme forms miRNA?
4). what does miRNA form a compplex with to become active? what is the functional component
1). short- a 30 NTs long= arise from non coding regions or introns, when spliced out become active
2). Drosha = makes just a BP- loop stru ture of pre-mrna = transported into the nucleus
3). Dicer = dices off loop, now just complimentary miRNA strands
4). forms a complex with risc = degrades miRNA* strand = leaves functional miRISC
1). what happens to mRNA when miRISC has perfect complementary binding
2). incomplete complimentary?
1). perfect = DS break, unprotected ends, and mRNA is degraded
2). no cleavage, but doesnt not translated.
1). how are these produced?
2). what enzyme processes dsRNAs?
2). what do they form complexes of?
4). how do these interfere with gene expression?
1). a gene is transcribed in both directions = creates complimentary rnas that hybridize = dsRNA. processed to make small dsRNA pieces
3). ribonucleoprotein with argonuate
4). can selectively block or interfere with expression of genes with complimentary regions, destroy viral mRNAs
1). what are these produced from? what do they interact with ?
2). what do piRNAs make complexes with?
3). what does the complex degrade? what does this prevent?
4). how can this complex also modify TE transcription epigenetically?
1). produced from piRNA genes throughout genome = interact wuth expression of transposable elements
2). form complexes with PIWI proteins (similar ish to risc)
3). degrades transposable element RNAs to prevent transposons from integrating in genome
4) modify histones areound TE genes, block dranscription
1). what does it mean to clone DNA, cells, organisms.
2). what are restriction endonucleases?
3). what to they recognize? is it in an isolated spot anywhere in the genome? what is a common defining feature
4). are blunt or sticky ends preferable to produce restriction fragments? what are the function of them?
1). generate/produce identical copies
2). enzymes that recognize specific sequences in the dsDNA and cleave the DNA
3). recognize a specific set of sequence of bases in DNA; palindromic = top strand and bottom strand read the same 5'->3'
4). sticky - can use these 3' SS overhangs to allow recombination later on = brings DNA from two different sources
1). what is the equation to calculate average fragment length?
2). how do you calculate how many fragments would theoretically be made in the genome with 3 bil BPs
1). 4^n (n = bases in recognition site)
2). 3 bil / fragment length from above
1). if circular dna, # restriction sites = ________ fragments
2). if linear dna, # restriction sites = ________ fragments
3). how are DNA fragments produced?
4). on gel electrophoresis, why does DNA travel toward the end of the plate? what does distance travelled show you?
1). restriction sites = fragments
2). restriction sites = fragments - 1 (there are one more fragments than restriction sites
3). run on gel electro no enzymes, different enzymes, single adn double digests to figure out wheere its being cut
4). end of plate is + charged, DNA is - charged and is attracted; short distance = large; long distance = small
interpreting restriction map
1). what does undigested lane tell you?
2). if a lane has two bands but a single digest, what does this show you?
3). if a lane has multiple fragments and a double digest, what do the bands show you?
4). how do you use this to interpret order of restriction sites?
1). the total length of DNA
2). shows two fragments =the size of both
3). double digest, tells you the sizes of all three fragments
4). if EcoR1 makes 1000 and 600, theres one cut
bam makes 1400 and 200
together = 600, 800, 200
bam makes a 200 and 1400, so bam is at 200 end. ecor1 is at 600 = 800 between them.
1). what is a cloning vector?
2). what is the function of origin of replication in plasmid? can you have more than one
3). how do restriction sites insert foreign DNA of choice?
4). why is anitbiotic resistance chosen as a marker?
1). a plasmid or virus that carries foreign DNA to replicate it.
2). used to initiate replication, can include prokaryotic and eukaryotic so can be replicated in both
3). restrictions ites are used to open u the DNA
4). then treat bacteria with antibiotics to kill out the cells that didnt take up the plasmid = easy purification
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