Dr. Bruist – Molecular Biology – Exam III – Protein Synthesi – Flashcards

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Anticodon in the anticodon loop reads what on what?
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The codon on the mRNA.
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How does the Amino Acid attach to the tRNA?
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It is esterified to the 3' end of the tRNA at Cytosine-cytosine-adenine end (or CCA stem).
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What are some commonalities between different tRNAs? Why?
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They have all the exactly same structure because they need to fit into any ribosome the same way.
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What puts amino acids with their appropriate tRNA?
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Aminoacyl-tRNA synthetases
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What is the name of the complex made by amino acids and their tRNA?
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Aminoacyl-tRNA -> Covalent compound
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For each amino acid there is a unique...
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Aminoacyl-tRNA synthetase to bind it to tRNA.
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Aminoacyl-tRNA synthetase uses...
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ATP to make a high energy ester linkage between the amino acid and the tRNA.
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Ribosomes are usually...
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70S.
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Large subunit is usually..
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50s = 23s + 5s RNAs + 34 proteins
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Small subunit is usually...
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30s = 16s RNA + 21 protiens.
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What are the three sites for tRNA?
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APE sites
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What is the A site?
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The A site is for the incoming aminoacyl-tRNA group.
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The P site of Ribosomes are for the...
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Peptidyl site where the previous tRNA is attached to the growing peptide chain.
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The E site is for...
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...exit site where the deacylated tRNAs leave.
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In what way are amino acids synthesized?
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They are made amino to carboxylic end.
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mRNA is read by the ribosomes in what order?
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5' to 3'
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Which part of the mRNA is translated?
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The ORF (Open reading frame)
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How many ribosomes can bind to mRNA?
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Many -- its hard to count.
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Can the ribosomes catch mistakes if the wrong amino acid is attached to the tRNA?
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No.
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What is the initiator codon in prokaryotes? How is it recognized?
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AUG. It is recognized by an initiator tRNA.
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In prokaryotes, the initiator sequence AUG is the first AUG after what?
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The Shine-Delgarno sequence.
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What is the Shine-Delgarno sequence?
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The Shine-Delgarno sequence is site where the ribosome binds onto the mRNA.
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The Shine-Delgarno sequence interacts with which part of the rRNA?
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16s part of the small subunit of the RNA.
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In prokaryotes, the initiator tRNA carries what?
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N-formylmthionine (fMet)
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In prokaryotes, there are a bunch of initiation factors which recruit...
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...mRNA, small ribosomal subunit, and the initiator fMet-tRNA complex.
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The large subunit is added when?
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After the addiction of the small subunit.
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After the addition of the large subunit, what gets added next?
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fMet-init-tRNA is in the P site. The A and E sites are open.
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What type of hydrolysis is uded to make sure that prokaryotic initiation is correct?
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GTP hydrolysis.
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In prokaryotes, which elongation factor brings in the new aminoacyl-t
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Tu factor elongation is checked by what?
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GTP hydrolysis double-checks codon and anticodon pairing.
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After the peptide bond forms, where does the growing peptide move to?
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It is transferred to the tRNA on the A site.
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In Translocation, where does everything go? Which elongation factor mediates this? E site tRNA eventually goes where?
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The A site tRNA goes to the P site. The P site tRNA goes to the E site. Elongation factor G mediates this process while hydrolyzing GTP. The deacylated E site tRNA leaves the ribosome.
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In prokaryotes, how is termination recognized?
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Release factors recognize stop codons on the mRNA. Triggering termination, they cause the release of the grown peptide to water, rather than the amino acid of another tRNA.
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In order to keep translation accurate in prokaryotes, which biochemical opathway is used to insure it?
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GTP hydrolysis
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In prokaryotes, where are the three ways in which GTP hydrolysis is used?
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Initiation is at the right place so the right ORF is used.
The correct tRNA matches a codon.
The ribosome advances exactly three codons to prevent a frame shift.
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In Eukaryotes, the ribosomes have size of what?
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80s = large subunit is 60s + small subunit 40s.
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What is different about the subunits between eukaryotic and prokaryotic translation?
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Eukaryotic subunits are bigger and have larger RNAs. The large subunit has an extra rRNA.
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In eukaryotes, where does initiation start?
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The ribosome loading site is the cap on the 5' end. The poly A tail also somehow helps in loading.
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What is the initiator sequence?
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It is the the first AUG sequence seen by the ribosome coming in from the 5' cap.
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In eukaryotes, how is the intiator tRNA different from prokaryotic initiator tRNA?
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The initiator tRNA is not not formylated. It only contains methionine.
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Because eukaryotic and prokaryotic translation is different...
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...drugs can be used as antibacterial agents which target protien synthesis. (Erythromycin & Azithromycin).
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After the protein is made, what's next?
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Targeting: we need to figure out where the protien belongs.
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Information for protein targeting is where?
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Its read from the amino acids of the protein as it emerges from the ribosome.
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Explain the way the protein is secreted?
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The N-terminal sequence initiates the binding of the SRP - signal recognition particle.
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After the SRP binds to the ribosome, what happens?
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The ribosome and the growing protein are brought to the ER so that it can be direectly released into the ER.
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Protein enter or cross the membrane where?
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At the ER
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What does the ER do?
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It does post-translational processing.
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Efficient Metabolism and differentiation require what?
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Require control of gene expression.
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What are the 2 kinds of transcriptional control?
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Negative control - repressors prevent transcription.
Positive control - activators stimulate transscription
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Operon
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Group of genes that are under control of the same regulatory system
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Promoter
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transcription start site for RNA polymerase
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Operator
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a sequence of DNA to which a regulatory protein binds
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Repressor
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a protein that binds to the operator to prevent transcription
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Inducer
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a small molecule which makes the repressor leave the operator.
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Activator
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A protein that binds to the operator to enhance transcription.
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Co activator
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A small molecule that causes an activator to bind to its DNA site
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The lac operon is an example of which type of gene control?
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It is prokaryotic gene control.
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How many protein does does e.coli have to make in order to metabolize lactose?
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three
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Which questions will be asked when the production of lactose metabolism is going to occur?
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Q1: Is there any lactose around for these enzymes to use?
Q2: Is there a better energy source (glucose) available?
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For determination of lactose usage - Q1 needs to be answered _____. Q2 needs to be answered _______.
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Q1 needs to be answered yes. Q2 needs to be answered no.
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What are the three gene loci for lactose operon?
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lac z, lac y, and lac a
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What does lac i code for on the lactose operon?
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Lac i translates the repressor protein.
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What is the CAP protein in the lactose operon?
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A global activator whose action is linked to glucose availability.
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Which protein is associated with answering of the first question of lactose operon?
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The repressor formed from the lac i.
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In the absence of lactose what happens to the attached repressor of the lactose operon?
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The inducer is not attached, and so the the protein is in its repressing conformation: it binds to DNA.
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What happens when lactose is availiable in regards to the repressor protein on the operon?
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The inducer is made and attaches to the repressor. This changes the conformation of the repressor and causes the repressor to be released from the operon.
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What are the names of the actions done in answering the first question of the lactose operon?
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Repression and Induction
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What is question 2 of the lactose operon?
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Is there a better sugar (like glucose) availiable for fuel?
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What is the name of the action used to answer question 2 of the lactose operon?
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Positive control
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When lactose is absent, the repressor keeps what off the operon?
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It keeps RNA polymerase off the operon the DNA - which stops transcription.
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Describe the binding mechanism of repressor. How does it affect timing of attachment and release?
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It has cooperative binding - it attaches on fast and is released fast.
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What does CAP stand for?
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Catabolite Activator Protein
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What is the master element of the positive control of lactose operon?
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The CAP protein
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What does the CAP protein do when there is no glucose in the cell?
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It aids RNA polymerase in recognizing the lac promoter.
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How is the activity of CAP regulated through?
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Throught the presence of cAMP.
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The default setting of most eukaryotic genes is...
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..off.
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What interferes with gene expression when it comes to structure of chromatin?
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The histone-DNA interactions of the nucleosomes can interfere with translation.
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In order to loosen the DNA-histone interaction, what needs to be done?
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The histone needs to be acetylated.
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What acetylates the histone?
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HAT - histone acetyltransferase.
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What are the three things that need to be done to activate the euchromatin? (make it ready for transcription?)
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1.HAT acetylates histones
2.Remodeling engine moves histones.
3.RNA polymerase, transcription factors, activators and mediators attach.
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Activator proteins bind to what on the DNA?
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They bind to enhancers: DNA sequences.
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Do enhancer-activator complexes act at a distance or locally?
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Act at a distance.
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What are all the different locations that an enhancer location can be?
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It can 5' to the intron (upstream) or 3' to the intron (downstream). It can also be found within the neuron.
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The enhancer-activator complex binds to what?
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It binds to the mediator and the preinitiation complex.
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By the binding of the enhancer-activator complex to the mediator and the preinitiation complex, what happens to the DNA strand?
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It becomes looped over itself.
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What DNA-protein complexes limit the action of enhancer-activator complexes?
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Insulators
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What is a multicomponent enhancer-activator complex?
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Enhancesome
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The Interferon Beta genes has how many kinds of peptides and how many overlapping DNA?
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5 kinds of polypeptides and 8 overlapping sites
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Eukaryotic regulatory proteins can be multifunctional. Which means?
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They can act as an activator, repressor of transcription, or repressor of translation.
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Whats the significance of needing multiple activators for one promoter?
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It offers combinatorial control; which means that we can use a different combination the enhanceosomes to turn on different genes.
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Can a gene have multiple promoters?
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YES.
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Activators can be modulated by what else?
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Hormones like estrogen.
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On the activator - are there separate sites for DNA and estrogen binding?
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Yes. Estrogen acts as a modulator for activator protein.
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When estrogen attaches to the activator protein, what happens to the activator protein?
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It goes through a conformational change which opens the active site for a coactivator to attach.
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What are homeotic genes?
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Genes that are controlled by one entire developmental pathway. Mutations of this can cause tissues to become another type of tissue.
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What is a segment?
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It is the basic unit of the body plan of insects.
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Segment formation in fruit flies is determined by what type of gene group?
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Homeotic genes
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Which gene determines the front of each segment?
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The eve gene.
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What do reporter genes do in regards to the expression of the eve gene?
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They let you know which of the enhancers have activators attached to them.
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What was the reporter gene used for testing activated enhancers of the eve gene?
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Beta - galatosidase gene
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At stripe 2, the eve gene has activators. What are they?
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Bicoid and Hunchback
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At strip 2, the eve gene has repressors. What are they?
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The repressors are Kuruppel and giant.
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How do the repressors repress the eve gene?
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They keep the bicoid and hunchback from binding.
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What are the connections of fruit flies to mammalian genes?
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They share homeotic genes.
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What are the group of homeotic genes that are similar in arrangement between mice and fruit flies?
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It is the HOX genes. They control the anterioposterior arrangement of the spine.
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TOPO cloning is type of way to
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to put DNA fragments into a vector without the use of DNA ligase.
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TOPO cling ususally has DNA fragments that were amplified by which two types of polymerases?
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Taq and Pfu.
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In TOPO cloning, what is the most important enzyme that breaks the vector and puts allows the PCR products to attach?
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DNA topoisomerase 1.
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DNA topoisomerase 1 causes the vector to have what type of ends?
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5'-(c/t)cctt-3'
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Monoclonal antibodies are made from which two types of cells fused together?
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differentiated B-lymphocytes and cancer cells. They make a hybridoma.
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In random mutagenesis, what happens?
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The DNA is exposed to lots of polymerases and reactive substances to cause mutations.
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What do you use for site-directed mutagenesis?
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synthetic oligonucleotides.
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What are the 4 ways of getting genes into cells?
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Microinjection of germ cells, electroporation (transformation through an electric shock), transfection (lipid encapsulated DNA), and Viral transduction.
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If you designed a peice of DNA, what are two ways that you can insert them into the chromosome?
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Illegitimate (nonhomologous) recombination and General recombination.
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What is Illegitimate recombination?
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It is nonhomologous recombination between unrelated types of DNA. It introduce DNA anywhere in the chromosome. It is bad becuase uncontrolled but transposons make efficiency better.
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What is general recombination?
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This can deliver DNA to a precise location. The vectors are circular - just needs to match up the sticky ends.
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In ecoli, which promoter can we ascribe to certain genes to induce their transcription?
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We can use the lac promoter.
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tTA is the what to the Myc oncogene?
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It is the activator.
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When mice are treated with tetracycline, what happens to the tTA protein?
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It becomes inactivated - the tetracycline becomes a repressor.
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A displaced insulin enhancer/promoter can do what?
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It can turn on neighboring genes, but usually only in pancreatic cells.
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What can show succesful incorporation of genes?
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Selectable marker genes and reporter genes.
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Site specific recombination can reduce
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problems with verification parts of DNA, since its site specific.
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Transgenic Mice has two ways of breeding the gene of interest. What are they?
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Microinjection of germ cells. Embryonic Stem cell Injection.
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In the Microinjection of germ cells experiement with transgenic mice, what happens?
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The zygote is injected with the gene of interest. Than the zygote is transplanted into the womb. The progeny trnasgene shown through PCR fragment.
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In embryonic stem cell injection, what happens?
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The stem cells of a blastocyst is put into another blastocyst with original stem cells.
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The mice progeny that arises from having their stem cells mixed with another mouse's are called what?
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Chimeric mice. They are mosaics of the two original organisms.
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What is so good about transgenic mice?
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They allow scientists to knockout sertain genes - act as mouse models of genetic diseases.
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What does epigenetics do?
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We can change gene expression by not altering the sequence.
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What are three components of epigenetics studied in class?
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1.Gene dosage and X deactivation.
2.Imprinting
3.Environment ability to change epigenetic state.
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What are three mechanisms used in epigentics to control gene expression?
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1.Noncoding RNAs (wasted RNAs)
2.DNA methylation
3.Histone modification
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Males have one X chromosome, but females have two. How do Drosophila and C.elegans balnce this two?
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Drosophila doubles transcription from the male X. C.elegans halves the transcription rate of each X chromosome.
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In activated chromosomes in mammals are called
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Barr bodies
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The Barr resembles what?
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Heterochromatin
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When is the inactivated X chromosome chosen during development?
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It is chosen early in embryogenesis.
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How is the agouti gene controlled in mice?
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If methylated it represses expression, it can cause variegated expression of mottled gene.
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Heterochromatin is transscriptionally active or inactive.
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Transcriptionally inactive.
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X inactivation in mammals is similar to what we see in
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drosophilia
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In activations starts where on the chromosomes?
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It starts at the center and drops off with distance.
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What does Xist mean?
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X-inactive specific transcript.
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What does Xist do?
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It coats the Barr body, establishing inactivation. Some genes are still on though (Xist gene).
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If xist RNA is removed, is the Barr body still inactivated?
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Yes.
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What is the complement and opposer of Xist?
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Tsix
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What is imprinting?
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Imprinting is when certain genes behave differently because they were inherited from the mom or dad.
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What does igf2 stand for?
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Insulin-like growth factor.
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The paternal gene for Igf2 is
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downregulated. It is only expressed by the father's genes.
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The maternal gene for Igf2 receptor is
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downregulated. It is only expressed by the mother's gene.
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If the igf2 gene is lost then what happens?
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The baby and the placenta will be very small.
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If the loss of the Igf2 receptor leads to what?
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Large offspring.
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In the imprinting experiment, the transgene was inserted where?
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Into the immunoglobulin locus.
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How did insertion of the c-myc gene show imprinting?
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Only males expressed the gene, females repressed it through imprinting.
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How was the c-myc gene repressed through imprinting by the females?
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The gene was methylated.
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Methylation prevents what enzyme from doing what?
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It prevents restriction enzymes from cutting their sites.
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Maternally inherited transgenes are always...
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methylated, regardless of the methylation state of the mother.
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Paternal transgenes are always
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unmethylated. Regardless of the methylation state of the father.
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Artificail methylation can...
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turn off gene expression when needed.
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With what does histone deacylate?
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With a methyl-binding domain it deacetylates a methylcytosine-containing chromatin.
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Methyl transferases recognize what?
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CpG.
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What does methyl transferases do to CpG?
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It fully methylates them.
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What the problem with methyl-cytosine?
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It can deaminate to thymine. Mutations can result.
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Methylation can turn off...
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insulators - causes expression.
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Children of mother's who survived the hunger winter had what?
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Had different DNA methyaltion patterns whicj were different than their siblings.
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Changes in the imprinting patterns in twins are associated with...
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observable differences between twins.
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Rhodopsin is what kind of receptor?
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7TM receptor. G Protein Coupled receptor.
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