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223 Cards in this Set
- Front
- Back
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Terminators
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trigger the elongating polymerase to dissociate from DNA and release the newly made RNA chain
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Rho-Dependent is a what?
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Terminator
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Rho dependent requires what to terminate? and what does it do?
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Rho protein. it binds a single stranded RNA as it exits the polymerase and uses ATPase to pull it away from the DNA template and polymerase
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What two rules does Rho follow?
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It first bind Rut sites (that are not secondary structues) Then if a transcript is being translated (bound by ribosome) it will aviod that transcript
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The only initation factor bacteria needs?
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Sigma factor
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Initiation factors that Eukaryotes use?
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General transcription factors (GTFs)
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core promotor
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Minimal set of sequence elements required for accurate transcription by the RNA Pol II
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The TATA box is what? and is bound by? and how?
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Where it all binds. TFIID. (Transcriptional factor) Through its TATA binding protein (TBP)
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What does the TBP do?
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distorts the TATA box and recruits TFIIE and TFIIH and the mediator Complex
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What does TFIIH do?`
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provide the necessary helicase activity and uses ATP
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In orfder for the RNA Pol II to elgongate what must it do?
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Get ride of the TF. (once it starts elongating it will be leaving proteins behind)
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How does this happen?
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By phosphorylation of the C-Terminal domain (CTD) activated by a kinase found in TFIIH
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Which TF also has a kinase?
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TFIIH
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When transcribing Chromatin, activators do what?
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help recruit polymerase to the promotor, stabilizing it's binding site
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What does the mediator complex do during chromatin transcribing?
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Mediates the activators recruitment, and also bind the CTD of the RNA Pol II
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Yeast and human mediator complexes contain ? subunits. ? of which are homologous
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>20. 7
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Steps to elongation of Transcription
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Pol II sheds the initiation Factors and they are replaced with elongation factors.
CTD recruits factors for processing. (BRINGS IT ALL TOGETHER AT THE SAME TIME) |
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What processing factors are recruited by the CTD
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RNA capping enzyme on the end of the RNA
RNA Splicing machinery assembles next Then the cleavage and polydenylation factors. |
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What stimulates the bonding of these factors?
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The phosphorylation of Serine
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How does the RNA pol get past histones?
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Spt16/SSRPI work together to disociate the H2A-H2B dimer.
The RNA Pol then can roll through Then Spt16/SSRPI then reassemble the nucleosome with the help of SPT6 |
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What is the first modification of new RNA?
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It is capped at it's 5'
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How does this happen
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Removal of a phospahte at the 5' end
guanine is added Methyl group is added to the 7N position |
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Final modification of new RNA?
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Polyadenylation at its 3' end
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What is crucial for this polyadenylation at its 3' end?
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CTD
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Steps in the Polyadenylation : first step
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The enzymes cleavage and polyadenlyation specificy factor (CPSF) and the cleavage stimulation factor (CSF) move from the CTD of RNA Pol to the poly-A Sequence (at the begining of the new RNA)
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Second step
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Other proteins are recruitted and RNA is cleaved
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Third step
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CPSF binds the Poly-A signal and Poly-A Polymerase (PAP) is recruitted to add more adenines to the 3' end of the mRNA
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Fourth step
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around 200 adenines are added, poly-A binding proteons begin to bind the long stretch of poly-A
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Fifth step
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All poly-A recruitment factors and enzymes dissociate from the mRNA
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LECTURE 16
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LECTURE 16
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exons
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coding
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introns
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non coding
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Difference between pro and eukarytotes when it comes to gene structure?
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exons are interupted by introns in Euk
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transcription of eukaryotic genes produce?
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pre mRNA
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After the pre mRNA is produced, the ? are removed by ?
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Introns. RNA splicing
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exons longer or introns longer?
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introns
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How does it relates to complexity?
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More introns, more complex. more introns make the organism more diverse bc there is more control in splicing
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intron-exon boundary
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site that will be spliced
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Branch point. what is it, found where.
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point where the spliced 5' introns makes the lariet. entirely in the intron
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First step in splicing
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the 2'-OH A site attacks the Phosphate at the G site.
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what was that result?
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the freed 5' end of the intron is joined to the branch site
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Second step
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the 3'-OH group of the recently freed exon attacks the phospahte at the 3' splice site
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The result
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the two exons are spliced together and it releases an intron lariet
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trans-splicing
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splicing between two separate exons
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what happens?
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same mechanisms, only a Y shaped structure is produced
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What mediates the trans-esterfication rxns? and whats its major functional unit?
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Splicesome. RNA
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RNA has how many and what are they called
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five. U1 U2 U4 U5 U6. snRNAs
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Each of these is paired with several what? new name?
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Proteins. snRNPs
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what does U1 do
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labels the 5' splice site
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U2FA?
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binds the 3' splice site
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exons
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coding
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introns
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non coding
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Difference between pro and eukarytotes when it comes to gene structure?
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exons are interupted by introns in Euk
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transcription of eukaryotic genes produce?
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pre mRNA
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After the pre mRNA is produced, the ? are removed by ?
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Introns. RNA splicing
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exons longer or introns longer?
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introns
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How does it relates to complexity?
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More introns, more complex. more introns make the organism more diverse bc there is more control in splicing
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intron-exon boundary
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site that will be spliced
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Branch point. what is it, found where.
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point where the spliced 5' introns makes the lariet. entirely in the intron
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First step in splicing
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the 2'-OH A site attacks the Phosphate at the G site.
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what was that result?
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the freed 5' end of the intron is joined to the branch site
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Second step
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the 3'-OH group of the recently freed exon attacks the phospahte at the 3' splice site
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The result
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the two exons are spliced together and it releases an intron lariet
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trans-splicing
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splicing between two separate exons
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what happens?
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same mechanisms, only a Y shaped structure is produced
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What mediates the trans-esterfication rxns? and whats its major functional unit?
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Splicesome. RNA
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RNA has how many and what are they called
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five. U1 U2 U4 U5 U6. snRNAs
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Each of these is paired with several what? new name?
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Proteins. snRNPs
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what does U1 do
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labels the 5' splice site
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U2FA?
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binds the 3' splice site and helps the branch point binding protein (BBP) bind to the branch site
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What replaces the BBP and binds to the branch site and extrudes (unpairs) the A site?
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U2
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What three are then added? and what do they do?
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U4 U5 U6. pulls together and produce lariet
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which two are held closer together?
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U4 and U6
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which one leaves now and what replaces it? and why?
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U1 leaves because it's only job was to define the boundary and U6 has pulled the link together and replaced it
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who does the annealing?
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U6
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what happens when U4 leaves?
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U2 and U6 can now bind
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what happens when U2 and U6 bind?
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the form the active site and break the 5' end and attaching it to the branch site
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which helps to bring the two exons together?
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U5
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Most common spilcing?
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pre mRNA splicesome (with the spliceosome
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2nd most common?
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Group II self spilcing. same mechanisms, doesn't need a spliceosome.
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Least common splicing?
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Group 1 self-splicing. uses secondary structure to fold the RNA
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What is it called when the exon sites are skipped?
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exon skipping
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pseudo splice sites
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when other close splice sites are mistakenly read as splice site and it is cut in half
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loading on splicing machinery during transcription would be one way to improve...
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the accuracy of splice-site selection
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What do SR proteins do?
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bind to exonic splicing enhancers (ESEs) within the exons
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What does this do?
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The SR proteins bound to the ESEs interact with the splicing machinery and recruit it to nearby splice sites.
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SR proteins are responsible for recruiting which of the U1 U2 U4 U5 U6 proteins?
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U1 to the 5' and U2AF to the 3'
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LECTURE !7
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LECTURE 17
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degeneracy
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amino acids are encoded by more than one codon
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synonyms
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codons that encode the same amino acid
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mutations in the first nucleotide positions will often,,,
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will give the same amino acid
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pyrimadines in the second position code for ...
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hydrophobic amino acids
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purines in second position encode for
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polar amino acids
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Transition mutations in the third position ...
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rarely changes an amino acid (50%)
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highly purified tRNAs can
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bind several different codons
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Which anticodon can bind the most codon bases?
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Inosine (3)
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which end of the anti codon is not as restricted?
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5'
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this allows it to?
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form hydrogen bonds with several different bases at the 3' end of the codon
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the codon is in the ? direction
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3'-5' direction
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codons are read by ribosomes in the ? direction
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5'-3'
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Codons are ? and mRNA contains ? gaps
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non-overlapping. zero.
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mRNA is translated in a ? from the ?
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fixed reading frame. start (AUG). meaning since they don't overlap, the nucleotide can be translated in any of the 3 reading frames
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Missense mutaion
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changes a codon specific for one amino acid to a codon specific for another amino acid`
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effect length of poly peptide?
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no
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nonsense (stop) mutation
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changes a codon from one amino acid to a stop codon
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effect polypeptide length?
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yes. makes it shorter than it would have been.
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Frameshift mutation
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where one or a small number of base pairs are inserted or deleated and alter the frame
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effect polypeptide lenght?
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maybe. you could get a stop codon earlier down the road
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Two ways to reverse mutations?
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reverse back mutations / suppressor mutations
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Reverse back mutaions
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where an altered nucleotide is changed back to its original arrangement
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suppressor mutations
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muatations that occur at different locations on the chromosome that supress the change
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Two kinds of suppressor mutations
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Intragenic and intergenic
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Intragenic suppression pg. 534
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mutations occuring in the original gene, but at a different site in the gene
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Intergenic suppression
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mutation occurring in another gene?
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Where the coordinated action of over 100 proteins and RNAs yeild a single protien
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Transcription
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Translation machinery has four compnents
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mRna
tRNA Aminoacyl tRNA synthetases Ribosome |
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mRNAs
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provide the template codons that specify the order of amino acids
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tRNAs
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the physical inteface between the mRNA and the added amino acids
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Aminoacyl tRNA synthetases
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couple the amino acids to the proper tRNA
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ribosome
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Composed of protein and RNA. (snRNSPs) coordinates recognition of the mRNA by tRNAs and forms the peptide bond between individual amino acids and the growoing polypeptide chain
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Open reading frames
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non overlapping stings of codons that secify different protein sequences
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What is a real ORF>
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Has the longest strain, with both a start and stop codon
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polycistronic mRNAs? Pro or euk?
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Have multiple ORFs and encode for more than one protein.
PRO |
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In PROs , in order for translation to occur what has to happen first?
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A ribosome must be recruited to the mRNA
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How is it recruited?
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Through the Prokaryotes Ribosome binding site (RBS)
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Where is the RBS located?
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upstream of the start codon
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Difference in Eukarytoes?
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the mRNA can be modified to help direct translation without the RBS
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How?
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capping on the 5' end recruits the Ribosome
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Start codon is also called?
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5'-AUG-3'
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Where is the Kozak sequence? what does it do? Pro or Euk?
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Before the start. increases translation efficiency. Euk
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Major difference between Pro and Euk transcription and translation
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Transcription and translation happen in the same place in Prokarytoes and not in Euk
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Where does transcription happen in Euks?
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Nucleus
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Where does translation happen in Euks?
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Cytoplasm
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Is translation faster in Pro or Euks? why?
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Pros, because it all hapens in the same place
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How does transcript/lation happening in the same place help make Pros go faster?
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Because the ribosome can procede with translating the mRNA as it emerges from the RNA Pol and multiple ribosomes can translate off the same mRNA
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DNA Pol does the...
Ribosomes do the... |
Transcription
Translation |
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The two subunits the compromie the Prokaryotic ribosome
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50s (large) subunit
30s (small) subunit |
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which contains the peptide transferase center, and what does it do?
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50s. it is responsible for forming the peptide bonds
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Which contains the decoding center, and what does it do?
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The 30s. where tRNAs read the codon units of the mRNA
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What is the in tact Prokaryotic ribosome called?
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70s Subunit
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What are the two subunits of a Eukarytoic ribosome?
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40s (small)
60s (big) |
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The intact Eukaryotic ribosome is called the
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80s
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What form of RNA is present in Ribosomes?
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rRNA
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HOw is the mass of each ribosome split?
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Between RNA and proteins
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More RNA or Proteins? why?
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More proteins because they are smaller.
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First step of Ribosome association
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mRNA binds to the 30 subunit first followed by the initiator tRNA
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second step
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the 50s subunit is recruited
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third
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protein synthesis is started at the 5' end of the mRNA
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fourht
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new tRNA are added and released to grow the poly peptide
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fifth
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the ribosome encounters a stop codon
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sixth
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the polypeptide chain is released
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7th
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the ribosome disocciates back into a large and small
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how many ribosome can translate the same mRNA?
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many!
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Why is this important?
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cells do not have abundant mRNA (1-5% of a cells RNA)
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What are the two most abundant forms of RNA?
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rRNA and tRNA
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New amino acids are attched to the gorwin poly peptide chain where?
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At the C-Terminus (Carbon)
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What is the single chemical reaction that ribosomes do?
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form a peptide bond between each new amino acid and the growing peptide chain
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the two charged species of tRNA that help addition of new amino acids
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Aminoacytl-tRNA
Peptidyl-tRNA |
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First step in the addition of new amino acids
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the peptidyl is present at the C-Terminus of the polypeptide
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second
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the amino (N) group of the aminoacyl-tRNA is brought close to the C-Terminus of the peptide-tRNA
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third
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the amino (N) group of the aminoacyl-tRNA attacks the C-terminus of the peptide-tRNA
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the result?
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and new peptide bond is formed
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proteins must grow from the ? to the ? termini
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N to the C termini
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Peptide transferase reaction
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the name of this whole process of the peptide bond being transfered from pep-tRNA to Amine-tRNA
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How many tRNA binding sites does the Ribosome have? and there names
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3. E, P, A
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The P site
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is the binding site for peptidyl-tRNA
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the A site
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the binding site for aminacyl-tRNA
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the E site
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the binding site for the tRNA that is released
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What is the order that tRNA binds these sites?
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P, A, E
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LECTURE 19
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LECTURE 19
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the tRNA that binds the P site first is called what?
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fMet-tRNA
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why is it called fMet-tRNA
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because it contains a formyl group (HCO)
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Most of translation occurs with or without a Full ribosome?
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Without, mainly just with the 30S
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There are three ? that help translation initiation
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initiation factors
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how are these initaion factors labeles?
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IF1
IF2 IF3 |
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what does IF3 do?
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occupies the E site of the 30s and prevents the 50s from binding
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what does the IF2 do?
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IF2 interacts with the IF1 and the initiator tRNA to help bind the fMet-tRNA
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How does IF2 help bind the fMet-tRNA?
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It uses GTPase
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What does IF1 do?
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Blocks tRNAs from binding the A site
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Step 1 of prokaryotic Translation Initiation
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IF3 bind the E site and blocks the 50s from binding
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second
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IF1 and IF2 bind and block the A site from binding tRNA
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third
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the initiator tRNA is recruited
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fourth
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it binds the Psite with the help of IF2
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fifth
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the mRNA start codon bind the initiator tRNA causing a shape change
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sixth
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IF3 is released and the 50s binds
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7th
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the creation of the 70s initiation complex causes the IF2 to hydrolysize its GTP causing IF2 and IF1 to leave
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8th
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Now with IF1 and IF2 gone, the A site can now bind new tRNA
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first Steps to Ribosome assembly in EUKARYOTES
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eIFA binds to the A site so stop the tRNA from binding to the 40s A site
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second
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eIF1, eIF3, eIF5 all bind the E site to prevent the 60s from binding
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third
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the eIF2/initiator complex is brought to the P site. the meth on this initiator tRNA is not special)
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fourht
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the 40s subunit is now recruited by the 5' capped mRNA
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First steos to mRNA assembly in EUKARYOTES
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5' cap is put on the 5' end of the mRNA
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second
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eIF4E recognizes the 5' capped end
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third
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additional initiation factors join in (eIF4G binds eIF4E and the mRNA)
(eIF4A binds eIF4G and then the mRNA) |
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fourht
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eIF4B binds and activates eIF4A helicase activity
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fifth
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the activated helicase causes eIF4A to unwind any secondary structes at the 5' end so that is can bind to the ribosome
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What is the name of the completed 40s ribosome combined with the mRNA?
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48S pre initiation complex
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REVIEW OF THE eIF's jobs
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REVIEW OF THE eIF's jobs
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eIFA
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binds the A site- prevents tRNA from binding
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eIF1, eIF3, eIF5
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all bind the E site and prevent 60s from binding
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eIF2/initiator complex
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binds to the P site
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eIF4E
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recognizes and bind the 5' caped end
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eIF4G
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binds eIF4E (at end) and eIF4A
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eIF4A
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binds eIF4G and the mRNA. Also uses its helicase do to the unwinding on the mRNA
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eIF4B
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activates the RNA helicase activity of eIF4A and it unwinds any secondary structures
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steps AFTER THE 48S preinitiation complex is formed
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the complex hydrolosizes ATP and scan the mRNA in a 5'-3' direction, looking for a start codon
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second
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the anticodon tRNA bind the mRNA start codon,
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third
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all the initiation factors are released and eIF5B comes in a hydrolysizes its GTP
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result?
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the 60s can now bind
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what does the poly-A tail do?
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keep the initiation factors closely associated with the 3' end of the mRNA and helps recycle ribosomes
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what does the eIF4G do?
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interacts with the poly-A binding proteins and circularize the mRNA
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what does EF-Tu do
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escorts aminoacyl-tRNA
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steps to do this
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EF-Tu binds the 3' end of the aminoacyl-tRNA and prevents it from binding until it is released
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EF-Tu can only bind aminacyl-tRNA when?
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When it is bound to GTP
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second
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EF-Tu-GTP takes the aminacyl-tRNA to the A site and binds it to the mRNA codon
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third
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EF-Tu interacts with the factor-binding center, causing it to hydrolsize its GTP and release from the aminacyl-tRNA
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1 stop to preventing incorrect aminoacyl-tRNAs to the codon
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two adjacent amines help from 3 bonds and these bonds are only the strongest when there is a correct pairing, if there is not one, they dissociate.
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2 way
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in order for the EF-Tu to release the aminayl-tRNA into the A sopt, the anticodon and the codon just match up, if they don't it will not release it
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3 way
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if an incorrect aminyl-tRNA is drop in the A site, when the amine roates to leave it's peptide, if it is incorrect it will just pop off. ACCOMODATION
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EF-G: steps to moving the tRNA from the A to the P
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EF-G muust first bind GTP to bind to the ribosome
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second
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During the hybrid state, the EF-G-GTP can bind the A site (factor binding site)
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third
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the A site stimulates the GTP and the EF-G extends down to the bottom of the A site and pushes it to the P site, causing the P site to over to the E
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fourth
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Now EF-G-GDP loses its affinity and is release, along with the old P site tRNA
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