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. |
