Please help me out with this biochemistry assignment. Thanks.
Please help me out with this assignment. Thanks.
2 W arm Up: 1) What is the sequence of the peptide pictured above in sing le letter amino acid code from N to C terminus ? (12 point s) 2) Dr aw a square around the planar at oms in one of the peptide bond s in the peptide . ( 4 point s) 3) Dr a w a rrows to a hydro gen bond dono r and a hydrogen bond acceptor in the peptide b ackbone. ( 4 point s) 4) W hat key principles allow ed scientists to correctly predict th at p roteins would fold into stereotypical s econdary structure of the alpha helix and th e be ta sheet? ( 8 point s) 3 5) Define the hydr ophobic effect, and describe how it contributes to the fo rmation of cellular membranes. ( 8 point s) 6) Why does paper lo se its structural integrity when it becomes we t? ( 8 point s) 4 One of the learning objectives in the c lass was to teach you about the structure and funct ion of macromolecules so that you can go for ward and apply these conc epts to what ever particular question you are int erested in . In the following sectio ns, we will analyze s ome experimental data in the context o f co ncepts we discussed in the class. DNA synt hesis Q 7 – 15 rel y on the following info: While nearly all organisms on Earth use a DNA code of ATCG, a particular class of bac teriophages (vi ruses that in fect bacteria) , was found to use ZTCG, where Z is an 2 amino adenine, a m odified fo rm of adenine . dZTP basepairs with thymine in the phage DNA, forming 3 hydrogen bonds. 7) Draw an AT basepair and a ZT basep air , inc luding the riboses and a phosphoryl group . ( 16 point s) 8) D raw an arrow to the additional hydrogen bond in ZT. (4 points) 9) Draw an arrow to a glycosidic bond . (4 points) 10) Draw arcs indicating the major and mino r groove s for both AT and ZT basepairs . (4 points) 5 11) How would you e xpect the substitution of Z with A in an oligonucleotide to change its melting temperature? Define melting temperature and e xplain two factors that will affect melting temperature. (1 2 points) 12) You synthes ize an oligonucl eotide with ATCG and with ZTC G and want to experimentally measure the melting temperature ( Tm ) using a spectro photo meter , which measures the absorbance of light at particular wavelengths , s u ch as 260 nm. Please draw a graph with an example of your expected data for two primers one with dATP and one with dZTP. Your graph should make cle ar the variables changed, the measurement taken, and how Tm can be derived from the resulting graph. What charac teristic of DNA does this t echnique take advantage of? (12 points) 6 13) The restriction enzyme P stI cuts DNA wit h ATCG and w ith ZTCG, while BamHI only cuts ATCG DNA. Describe how restriction enzymes can bind D NA in a sequence specific manner , and h ypothe s ize how thes e enzymes might differ in their recognition of DNA such that one is unaffected by the ZTCG subs titution. I suggest referring to you r dr awings in Q7 – 10. ( 8 points) Q 1 4 – 15 refer to the follosing information (and also refer to questions 7 – 13). To test the activity of each polymerase with dATP and dZTP, the researchers performed a primer extension assay with each pur ified polymerase where a fluorescently labeled primer (FAM is a fluorescent label) was annealed to a DNA tem plate such that extension required polymerization of either dATP or dZTP. 14) Given the data on k cat and K m above, which of the following gels for the primer extension assay corresponds the extension with the E. coli Klenow fragment and which to the Acine to bacter phage polymerase ? Note that the first two lanes show the full length product and initial primer, and the lanes labeled 1 and 2 correspond to the 7 situations presented above (1 = extension with dATP, 2=extension with dZTP). Explain your answer with a discussion of the K m and k cat values provided above. (15 points) Gel A p olymerase 1 Gel B polymerase 2 15) The p rimer extens ion assays also con tained the following components. Discuss why they are important for the reaction: a) 20 mM Tris – HCl (pH 7.5) ( 5 points) b) 5 mM MgCl 2 ( 5 points) 8 Transcription 16) Does DNA polymerase III or RNA polymerase II have greater intrinsic fidelity during elongation ? Describe 3 of the mechanisms of ensuring correct informati on transfer , making clear which ones are use d by which polyme rase . D iscuss ho w fidelity correlates with the p ur pose of each molec ule. (15 points) Q17 – 2 2 refer to the inf ormation b elow: This is an image of a DNA se foot printing experiment testing how the small molecule IPTG effects the binding of a transc ription fa c tor to DNA. IPTG activate s transcription at promot ers containing th e consens us sequence pr esent in th is DNA frag ment. A: 76 basepair DNA fragmen t DNAseI B: 76 basepair DNA fragmen t DNAseI 0.5 uM transc ription factor C : 76 basepair DNA fragmen t DNAseI 5 uM transc ri ption fac tor D : 76 basepair DNA fragmen t DNAseI 0.5 uM transc ription factor IPTG E : 76 basepair DNA fragmen t DNAseI 5 uM transc ri ption fac tor IPTG 17) Dr aw an additional lane next to lan e A showing your pr edicted result if the scientist ha d NOT added DNAseI to lane A. (4 points) 9 18) In your own words, exp lain how a DNA foot printing experiment wor ks . (10 points) 19) Binding of this tr anscription facto r (TF) to the 76 basepair DNA s equence can be described by the equ ation: DNA:TF à DNA TF a) Write th e equation fo r the K D (6 points) b) In l ane B, the total concentration of the transcription factor is 0.5 uM, the total concentration of the DNA is 0.1 uM, and the fraction of DNA bound by the tra nscription factor is 0.7. Using these numbers and the eq uation above, c alculate the K d . Please show your wo rk for part ial credi t opportuni ties. (8 points) 10 20) The small molecule IPTG ac tivates tran scription from p romoter regions contain ing this DNA sequence. Looking at the foot printing e xperiment, i s the transcription factor an acti vator or a repressor ? Given the data above d escribe a model for how IPTG activates transc ription. (8 points) Q 21 22 refer to t he informat ion below (and also the previous information for Q 17 – 23. Seeing the DNAseI foo tprin ting results, you set up an electrophoretic mobility shift assay with th e transcription factor and a 203 basepair D NA s equence from this promot er , and get the following pattern of your labeled DNA on a gel . 21) When running an electrophoretic mobility shift assay, do you want to add to SDS to denature yo ur protein? Exp lain. (1 – 2 sentences) (6 points) 22) You w ere expecting to see one band for free DNA and on e band for transc ription factor bound DNA. Come up with a hypothesis for why you are seeing more than th ese two bands on this 203 basepair fragment and descri be on e piece of evidenc e that would support your hypothesis. ( 2 – 3 sentences ) ( 8 points) 11 Q 23 27 refer to the follo wing information: Translation T his figure shows the rate of accumulation of inorganic phosphate ( P i) resulting from the hydrolysi s of G TP by E F – T u , as catalyzed by binding to the ribosome. The curve labeled Phe correspon ds to EF – Tu – [ g 32 P]GTP – Phe – t RNA Phe which perfectly match es the mRNA codon in the A site of the riboso me ( cognate ) , and Leu correspon ds to EF – Tu – [ g 32 P]GTP – Leu – t RNA Leu , which does not match the mRNA codon bound in the A site of the riboso me ( non – cognate ) . T he c alculated rate o f hydrolysis is 37/sec for the cognate tRNA and 55/sec f or the non – cognate tRNA. 23) The si te of co don: anticodon basepairing in the A site of the ribosome is appro xi mately 60 Angstrom s aw ay from the site of G TP hydrolys is in EF – Tu . Describe how the correct basepairing can cat alyze G TP hydrolysis. In your answer, please define the terms allostery and induced fit . (4 – 5 sentences) (1 4 poin ts) 12 24) The data above help ed d e fine the model of k inetic proofreading contrib uting to the fidelity of translation at the ribosome . Desc ri be t he concept of kinetic proofreading in your own w ords and how the rate of EF – T u mediated G TP h ydrolysis contributes to fidelity in translation. (3 – 4 sentences) ( 1 4 points) 25) From t he figure above , the ra te co nstan ts for GTP hydrol ysis were cal cul ated a s 37/sec for the cognate EF – Tu – [ g 32 P]GTP – Phe – t RNA Phe and 55/sec f or the non – cognate EF – Tu – [ g 32 P]GTP – Leu – t RNA Leu . a. Would you expect the rate constant for EF – Tu – [ g 32 P]GTP – L eu – t RNA Phe to be closer to the cognate rate of 37/sec or non – cognate rate of 55/sec ? Exp lain. (1 – 2 sent ences) (6 points) b. Would you expect the rate constant for EF – Tu – [ g 32 P]GTP – Phe – t RNA Phe to be closer to the cognate rate of 37/sec or non – cognate rate of 55/sec if the mRNA codon in th e A si te was switched from UUU to UU C? Explain. (1 – 2 sent ences) (6 points) 13 26) EF – T u mediated GTP hydrol ysis depends on His tidine 84. The pKa of free histidine i n solution is 6 . Calcula te th e pe rcent of histidine (the amino acid ) that is prot onated at pH 6 .5 and pH 8 .5 . Pl ease show your work. (1 0 points) 27) The figure below shows th e rate of EF – Tu m e diated GT P hydrol ysis from pH 6 .5 to pH 8 .5 . Make one hypoth esis about the role of His84 in the en zyme pocket of EF – T u . (8 points) You did it! Have a summer!