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107 Cards in this Set
- Front
- Back
What is needed for RNA transcription?
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-DNA template
-Ribonucleoside 5'-triphosphates -RNA polymerase |
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What is not needed for RNA transcription?
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A Primer
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What are the 3 major types of RNA?
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mRNA - 5%
tRNA - 15% rRNA - 80% |
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What are 2 other nonmajor types of RNA?
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siRNA (small interfering)
microRNA |
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What do microRNA's do?
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Bind mRNA to inhibit translation.
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What is snRNA? What does it do?
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Small nuclear RNA; it guides the slicing of the RNA endproduct.
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What key molecule is necessary for transcription?
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RNA Polymerase - it makes all 3 types.
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What are 2 forms of RNA polymerase can exist?
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-Core enzyme
-Holoenzyme |
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What is the RNA Pol core enzyme composed of?
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2 alpha
1 Beta 1 Beta' 1 w |
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What makes the holoenzyme of RNA Pol?
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The addition of a sigma unit.
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What is the core enzyme itself responsible for?
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Polymerization of the RNA molecule.
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Can the core enzyme operate by itself?
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No; without the sigma factor RNA Pol cannot recognize the promotor on the DNA template.
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What KIND of molecule is RNA polymerase?
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a Zn2+ containing metalloenzyme.
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what kind of linkage is made during RNA polymerization?
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The 3' OH of the RNA backbone nucleophilicly attacks the alpha phosphate of the incoming nucleotide.
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In what direction is RNA synthesized?
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5' to 3'.
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What is a major difference in the error rate of transcription vs replication??
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RNA polymerase has no proofreading capability in the 3'-5' direction so the error rate is higher.
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What is the coding strand?
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The nontemplate strand of DNA that can be used to decipher the primary AA sequence of the encoded protein.
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What are the 3 stages of RNA synthesis?
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1. Initiation
2. Elongation 3. Termination |
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What 3 things constitue an operon?
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1. Promoter
2. Operator 3. Structural genes |
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What is an operon?
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A genetic unit consisting of genes, and regulation regions.
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What is the lac operon?
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The genetic unit responsible for producing the enzymes that allow bacteria to utilize lactose.
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What takes place in the initiation phase of bacterial transcription?
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-RNA Polymerase holoenzyme binds the DNA template
-Scans DNA for promotor at -35 -Forms closed complex at -35 -Moves to 2nd site at -10 -DNA unwinds to form open complex at -10 -mRNA forms, sigma subunit is released |
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What kind of message is transcribed from the lac operon?
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a polycistronic message - three distinct gene products are produced.
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Where is the control region for the lac operon?
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Between the LacI and LacZ genes
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What is the difference between the closed and open complexes?
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Closed: RNA pol is stalled at the promotor region, but the DNA helix is still together.
Open: DNA melts apart and polymerization can occur. |
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What is the difference betweenthe -35 and -10 regions of the DNA template?
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At -35, the RNA polymerase holoenzyme simply binds to form the closed complex.
At -10, the DNA helix melts to form the open complex. |
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Why is the transition from closed to open complex the key event in DNA transcription?
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Because it exposes the start sequence on the DNA template strand intended for transcription.
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How is it that the DNA melts to facilitate formation of the open complex?
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Bacterial DNA is normally negatively supercoiled, so the unwinding process is favorable.
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What nucleotide do RNA chains start with?
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ppp-A or ppp-G; a triphosphate purine nucleotide.
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What happens to the sigma subunit after transcription initiation?
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It gets released as the polymerase moves on to the rest of RNA synthesis.
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Why is the sigma subunit so good?
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Because it reduces nonspecific binding of RNA Pol to DNA by a factor of 10^4 -> hence transcription only occurs at the correct promotor regions.
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What does the control regionof the lac operon consits of?
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CAP
Promotor Operator |
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What is CAP?
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Cyclic AMP Binding Protein
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What is consensus?
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The most common nucleotide at a specific sequence in genes from several different organisms. (e.g., -10 region)
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What is the consensus sequence at the -10 region in prokaryotic DNA?
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TATAAT
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Why aren't all -10 regions identical?
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Because there are different sigma subunits that are specific for each different -10 region.
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What is the result of deviation in consensus?
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Variation in affinity of the sigma factor for operator sequences; allows for different specificities.
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Why is it good for bacteria to have a variety of sigma factors?
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Their levels vary in response to environmental changes to help the bugs adapt.
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What are 3 examples of promoters recognized by different sigma subunits?
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Standard - TATAAT
Heat-shock - CCCATNT Nitrogen-starvation - TTGCA |
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How exactly does the sigma subunit change RNA Pol's interaction with promoters?
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-Increases specificity
-Decreases affinity |
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What would make a STRONG promoter? What would be the result?
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-Close conformity to the consensus sequence
-Result in frequent initiation |
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What happens during the Elongation phase of transcription?
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-Elongation proceeds 5'->3'
-Energy comes from phosphate cleavage of incoming nucleotide and PPi hydrolysis. -Multiple RNA Pol's sequentially bind promoter for continuous transcription. |
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When does the sigma factor dissociate?
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Immediately after elongation begins.
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What is the rate of elongation in E.coli?
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App. 30 nt/sec
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What facilitates formation of a transcription bubble?
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-Negative superhelicity in the DNA template.
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What does unwinding generate in the DNA being transcribed?
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Torsional stress from:
-Pos supercoils ahead of the bubble -Neg supercoils in its wake |
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How is torsional stress from transcription relieved?
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By topoisomerases
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What 2 antibiotics target topoisomerase II? What class are these drugs in?
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-Norfloxacin
-Ciprofloxacin Quinolone class |
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What are 2 drugs that interact with HUMAN topoisomerase?
What are they used to treat? |
-Doxorubicin
-Etoposide -Used for treating cancer |
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How do the topoisomerase-targeting drugs work?
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They ENHANCE the cleaving of DNA-double strands so much that it overwhelms the cell's ability to repair the damage.
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What antibiotic is used as an inhibitor of topoisomerases?
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Novobiocin
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How much DNA is unwound by each RNA polymerase molecule?
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17 nt per polymerase molecule.
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What are the 2 types of transcription termination that can occur?
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1. Protein dependent
2. Protein independent |
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What is the protein referred in "protein-dependent termination?"
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Rho
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What is a similar feature of both termination alternatives?
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Both depend on a hairpin loop structure.
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What is the Rho protein?
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An ATP-dependent helicase that binds nascent RNA, uses ATP energy to catch up to RNA Pol, and truncates the DNA/RNA helix.
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WHat helps the Rho protein catch up to the highly processive polymerase?
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-Txn bubble pauses as it hits a hairpin loop encoded in the DNA template.
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Where is the active signal for txn termination then?
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In the nascent RNA chain ..?
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What is the more common mechanism for Txn termination?
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The protein INDEPENDENT mechanism.
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What is a palindrome?
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A sequence of units (such as a strand of DNA) that has the property of reading the same in either direction
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How does a palindrome aid in transcription termination?
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As RNA is transcribed, 2 palindromic sequences are incorporated so that enough nt's are between them to allow a hairpin turn to form.
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What role does the hairpin turn play in protein-independent termination?
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It slows down RNA polymerase and causes it to pause.
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What happens when RNA polymerase pauses?
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The sequence of U's in the new RNA transcript that are bonded to complementary A's in the DNA template dissociate b/c the A-U bond is the weakest type possible.
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So protein independent termination is caused by:
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-Stable hairpin turn
-Pausing of DNAP -Dissociation of weak AU bonds |
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What stabilizes the RNA hairpin turn?
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It is rich in G-C, which are strong bonds.
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What is a-amanitin?
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a mushroom toxin directed at RNA polymerases in humans (euks)
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How many RNA polymerases are found in eukaryotic cells?
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3
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What does RNA pol type I synthesize?
Where is it found in cells? |
Ribosomal RNA - 18S, 28S, and 5.8S b/c it's in euks.
-Found in the nucleolus |
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What does RNA pol type II synthesize?
Where is it found in cells? |
mRNA precursors (immature)
snRNA -Found in the nucleoplasm |
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What does RNA pol type III synthesize?
Where is it found in cells? |
This mkes tRNA and 5S rRNA
-Found in the nucleoplasm |
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Which RNA pol types are inhibited by a-amanitin?
How strongly? |
II (strongly) and III (but only in high concentrations)
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So list 5 inhibitors of transcription:
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RADDC
-Rifampin -a-amanitin -Daunorubicin -DActinomycin -Cordycepin |
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What is rifampin's mechanism of inhibiting transcription? What disease is it usually for?
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Binds the B-subunit of bacterial RNA Polymerase to inhibit txn initiation.
-Used to treat tuberculosis. |
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What is the mechanism of a-aminitin?
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It is a potent inhibitor of RNA Polymerase II, the class that makes mRNA and snRNA.
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What is the mechanism of Dactinomycin?
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Intercalates between two G-C base pairs in DNA
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What is the mechanism of Daunorubicin?
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Intercalates between base pairs
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What is the mechanism of cordycepin?
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It lacks 3' OH so terminates the chain.
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What is the major difference in txn between Proks/Euks in terms of space/time?
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Txn is compartmentalized in Euks -> the RNA transcript must be transported from the nucleus before translation can begin. Poses a point for regulation.
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What is the eukaryotic promoter region?
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the TATA Box
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Where is the TATA box located?
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-30 upstream
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What prokaryotic sequence is the TATA box analogous to?
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The pribnow box (-10)
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What is the mechanism of Daunorubicin?
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Intercalates between base pairs
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What eukaryotic sequence is analogous to the prok -35 promoter sequence?
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The -80 CCAT sequence.
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What is the mechanism of cordycepin?
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It lacks 3' OH so terminates the chain.
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So what is the eukaryotic promoter region made up of?
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Two consensus regions:
-80 CCAT -30 TATA |
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What is the major difference in txn between Proks/Euks in terms of space/time?
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Txn is compartmentalized in Euks -> the RNA transcript must be transported from the nucleus before translation can begin. Poses a point for regulation.
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What are exons?
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Coding regions in eukaryotic monocistronic RNA.
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What is the eukaryotic promoter region?
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the TATA Box
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What are introns?
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Intervening regions that get excised from monocistronic RNA.
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What is the initiation event of eukaryotic txn?
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-Incorporation of the CAP structure.
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Where is the TATA box located?
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-30 upstream
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What is the CAP structure?
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7-methyl guanosine
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What prokaryotic sequence is the TATA box analogous to?
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The pribnow box (-10)
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What does 7-methyl guanosine consist of?
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-Methylated guanosine
-5'-5'triphosphate -2 riboses |
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What are the functions of the 5' CAP on mRNA?
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1. Enhanced stability of mRNA protected from nucleases
2. Enhanced translation efficiency |
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What eukaryotic sequence is analogous to the prok -35 promoter sequence?
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The -80 CCAT sequence.
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What enzyme is the CAP created by?
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Guanylyltransferase
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So what is the eukaryotic promoter region made up of?
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Two consensus regions:
-80 CCAT -30 TATA |
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What are exons?
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Coding regions in eukaryotic monocistronic RNA.
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What are introns?
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Intervening regions that get excised from monocistronic RNA.
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What is the initiation event of eukaryotic txn?
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-Incorporation of the CAP structure.
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What is the CAP structure?
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7-methyl guanosine
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What does 7-methyl guanosine consist of?
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-Methylated guanosine
-5'-5'triphosphate -2 riboses |
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What are the functions of the 5' CAP on mRNA?
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1. Enhanced stability of mRNA protected from nucleases
2. Enhanced translation efficiency |
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What enzyme is the CAP created by?
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Guanylyltransferase
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How are the riboses (sometimes) methylated in the 7-methyl guanosine CAP?
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By Methyl transferases, using SAM as a donor.
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What types of RNA are capped?
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Only one! mRNA.
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