CH339L.09.Test4a.Key-m - CH339L.09.Test 4 Name...

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Unformatted text preview: CH339L.09.Test 4 Name "‘4 (1; 35 points) Researchers have evolved a ribozyme that can perform a self—kinase reaction; that it is, it can transfer the gamma phosphate from ATP to a ribose. After allowing_the r_ibozyme to phosphorylate itse|f_(or.not)_,_ the—ribozymewasnrniiedwith _a _r_adiolal5e_l_e'<iol_igon_ucl_e_otide W*Primer, below) that bound to its 3’ end, reverse transcriptase ( , an deoxynucleoside triphosphates. RNA modifications such as 2’ phosphorylations will block the RT extension. After running a gel, the following results were obtained?“ '_ ' " (A) DNA *prirner size standard (B) *Primer + ribozyim (non-selfiphosp..mylated) + RT + dNTPs (C) *Priner + ribozyme (self—phosphorylated) + RT + dNTPs (D) Radiolabeled DNA size standard correspondirg to full—length ribozyme (a; 10 points) Based on this result, show 32‘. the diagram below approximately where the ribozyme was phosphorylated: [:1 Ribozyme 3’ i; __— (b; 15 points) Draw the ribose residue that was phosphorylated in atom.c detail, including any connections to other ribose residues in the chain. You rray represent the nucleobase connected to trie ribose as ‘N. r {r J’ ‘(T'm [6; 1D paints; “mu radihtatel the anew? unaapharyiated rheayme at its 5' and, and than partially {arachastieatyj hydrmyaa it Lain-g a strung base. then mn the pie-duets an a gel. Draw in the Expemed banda- InEfiCHn the gel bBlEIW lllllll||l|| {It} Full-length. radialabeted rihazyme {El} Nan-aelf-phdaphdrylated. partially hydraly'zed fibdryme [C] SeH-pheaahdwlated, partially hydrdhrzed ribdzyrne [El] Hajidlabeled DNA prirr-er arze standard [2; 15 pelnta]: A rad'relaaeled guaneama ia uaed ta initiate the aplte'rng eaaeada Shawn In Figure 25-15. The same nhdzyma cantinues the reaetiana ahdtrm in Figure 26-31(3). The radialahel is uttin'ratelz.r found in: {a} The 5' end at L-‘I'fl NS {h} The 3' end df L-TEI' l'u'S ‘3/The 5' end. at the "'15 nueledtrdea frdrn the 5' and" { The 3' and of the "19' nuc lafltidaa from the 5' and" {e} the a tree guanasina {f} A5 a free Qua ndaine 111:: haphaaahate ( (3; 25 points) In my lab, Randy Hughes has been attempting to engineer an orthogonal tRNA suppressor:tRNA synthetase pair not unlike those that are described in Box 27-3. He took a yeast tryptophanyl tRNA synthetase (WRS) and a yeast tRNA that had an anticodon engineered to bind to an amber (UAG) stop codon and introduced them into an E. coli strain containing a gene for green fluorescent protein that had an amber stop codon in it. He got the following levels of activity: GFP with stop codon: O RFU (relative fluorescence units) GFP with stop codon + yeast tRNA synthetase: 0 RFU GFP with stop codon + yeast tRNA synthetase + yeast suppressor tRNA: 1,000 RFU GFP with stop codon + yeast suppressortRNA: 100 RFU (a; 10 points) The most likely explanation for these results was: (L)\There was an endogenous E. coli suppressor tRNA in the organism already. fii)/An E. coli tRNA synthetase could charge the yeast suppressor tRNA. "(Ti/i) The yeast tRNA synthetase could charge an E. coli tryptophanyl tRNA. (iv) There was a translational frameshift mediated by the yeast suppressor tRNA. (v) The RNA was edited by the yeast tryptophanyl tRNA synthetase. (vi) The yeast suppressor tRNA is wobble pairing with other codons than the stop codon. (b; 5 points) Below draw the anticodon of a tryptophanyl tRNA, indicating 5’ to 3’ directionality. Then draw the mutation that must have been made in the suppressortRNA. You can use letters to represent nucleotides. (In), (Fig-if: C C 9*. r- /,._ . ., '} V k - F .. H.\ r ‘X—l a": \ {r .» w. r f l ' (c ; 10 points) Randy evolves the yeast tRNA synthetase to charge the suppressortRNA with a fluorescent amino acid known as Aladan. He adds the evolved tRNA synthetase and the yeast suppressor tRNA to an E. coli containing a gene that encodes a non— fluorescent protein (*not* the GFP gene) and that also contains an amber stop codon. He lyses the cells, runs the proteins on a gel, and finds fluorescence not only in his protein of interest but in many other proteins. The most likely explanation (that is also consistent with what you already know about this system) is: Aladan is charged onto E. coli tryptophanyl tRNA by the evolved yeast WRS. Aladan is charged onto E. coli tryptophanyl tRNA by the E. coli WRS. Aladan is charged onto the yeast suppressor tRNA by the evolved yeast WRS. Aladan is charged onto the yeast suppressor tRNA by the E. coli WRS. The editing mechanism of the evolved yeast WRS is now defective. (V) (4; 20 points) Below you will find a schematic of an array experiment. Answer the questions about this experiment. ORF = open reading frame, or gene. DNA micmarray making Micro scop e glas s slide 5 co ated with p olylysine + 6116 Yeast ORFs amplified by PCR l 5%? “““\ .‘Q‘Q‘I Sp utting (depu sit) Hyb rid'sau'nn Results deliver Scanning (lecture) uQQUIt... ‘£hl.t..s ‘Csyiur, l IIL‘II. Results analysis Stéplmm Le Cram DB 2003 (a; 10 points) This diagram is of course similar to Box 26-4 in your book. In your book it vaguely states that you “label cDNA with fluorescent dye.” Assuming you make cDNA starting from a poly(dT) primer, the green and red labels (Cy3 and Cy5) shown above were most likely added via: (i) Putting the labels on ribonucleoside triphosphates (e.g, Cy3-ATP) (ii) Purring the labels on deoxyribonucleoside triphosphates (e.g, Cy3-dATP) (iii) Putting the labels on an oligonucleotide that could hybridize to the 3’ ends of all cDNAs (e.g,, Cy3-some particular DNA sequence) (b; 10 points) The green / red ratio for a given spot that is quantitated in the “Results analysis" is indicative of: (i) The relative number of DNA genes between the two different strains. (ii) " The relative number of mRNA molecules between the two different strains. (iii) The relative number of DNA genes + mRNA molecules between the two different strains. (iv) The relative number of DNA genes x mRNA molecules between the two different strains. [5: 2D pair-rte) Eelew ycu will me a mention enemmete diagram for RNA pelymeraee {Ft} binding to a Dremeter {P} and firming e cleeed {Fin} and open [Fin]: eempIex. (a; 1D peinte} Tm: examine e dnfiereel pram-tater end flee the“ the eel}- rnereee forms a nureE'TetyeTEfl'Eee :fljfilgg'} but met the E u-f eeer: :emglex Eergnetier! 'Ie_mueh the same. fin an tne energyI stems end can Ieeting Imee er. tne dlegrem eta-eve fer this Seman premete-r. H, i. -' F..- j' ' (b; 1n peinte} Ultil’l‘IEl lely'r theLeLe_effi.eh—eEiE'ti-efi ENIIEHEHI eheujcl be re ughly preeertienel ID thfi ammunt Erf anrn complex formed {u 5 HI _ _ . For the eeeefid ere-meter yen described above ,r . :—'_‘“"_- {i} The rate ef Freeeerie’rim ir‘ifie’rim ie the eemn 3% W the 1‘ire’t Drummer. __1.. ii The “are n’ :raeeerirrl'l: .'| flit-erim 5'? +ele+er :ruen *r-a. f-re‘. meme-tee. IH] Tee We -_1" [mneeript-‘en inilretim I'e elewe ' Then .‘l‘E-f: first firemn’re'. [6; 3D petntefltj-Ethejnthetaee activity ie eften menitered by eeupling their reaetiene to a pyrepheephaE-eefiienflrem lueilfereee. When ATP and an amino acid are added to the eyntnetase. it creates purepheephate. which creates. right {er iumineseenee unite. LU}. Tau can also add tFt HA and then leak at the fermatien ef charged tFtHAs. “P? ~'i"'[:iu generate aeque nee euhetwliene in a leuey'l tRNfi. aynthetese and essay rt ueing either leueine er ieeleueiine aa eunetratea. "fa-u gel the fell-Ewing reeufl‘a: LU withtetl. Lllwithllia. Wehharged %tRMAL.eu charged with eu __-|' he tFthiA fir: tF-thie. with lie Mutant 1 “ tilt] LU '10!) LU tflfl‘t-i: -— — 1%— Mutant 2 1 DH] LU 1t“. LU meta 1.0% 1iI'I.|'iit:i-tg..ri:ie Lena LU I 1 LU watt-«3 1% (a: 15 paintle Which synthetaee has a mutatiari that decreaeee the Ku tdieeeeiatien cenetant} ef IEUC'yII tHMA eynfltetEBE Fer iE'.I:i|eI.J.ttine'?I {it Mutant 1 1'" t r T tiiiI I'u'ltnante Hath Mutant 1 anti Mutant}! [iv] Neither Mutant 1 nor Mutant 2 (tr: 15 paints-.1 'It‘u'hieh arnthetaae haa a mutat'hen that inhihita editing via hydrolysis of the aeylated tRHA? [ll Mutant 1 dfii‘ Mutant 2 {iii} Beth Mutahtt and Mutant 2 {w} Neither Mutant 1 net Mutant E ...
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This note was uploaded on 10/21/2009 for the course CH biochemist taught by Professor Kaplan during the Spring '09 term at University of Texas at Austin.

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CH339L.09.Test4a.Key-m - CH339L.09.Test 4 Name...

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