Critical Thinking Assignment Module 3 F2020 copy

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Critical Thinking Assignment for Module 3. Fall 2020 100 possible points NAME: Romello Bryant Due Date: Sunday October 18, 11:30 pm 1) In your own words, describe in less than 6 sentences what the graph below is showing. Provide information about each step, with respect to what is happening during PCR. 5 Points In the graph in the right it is showing each step in the PCR cycling process. In the first step the DNA strand is denatured by getting heated up to 94 ° c, where the hydrogen bonds of the complementary DNA strands break. After the first step, annealing takes place where Taq polymerase bind to the target DNA sequences and begins to initiate polymerization. Which we can see in the graph where in the second step the temperature is lowered down to 55 °c. Then in the third step of PCR Taq polymerase extends the primer, where new strands from the template DNA is duplicated. 2) Describe the process of transcription in your own words. Include the use these terms: RNA polymerase, DNA, promoter, unzipping and re-zipping, transcription stop site, and template strand. 5 Points In transcription, DNA in the gene is used as a template strand in order to make the messenger RNA, with the help of an enzyme which is called the RNA Polymerase. From there, three stages occur in transcription which are initiation, elongation, and termination. In the first stage which is initiation, the promoter functions as a region where RNA polymerase binds to the DNA strand making it the RNA polymerase binding site. During the biding stage, the double helix of the DNA strand begins to unzip and opens wide. Then in the next stage of elongation the RNA polymerase slides along the template strand, combining complementary nucleotides bases to the 3’ end of the messenger RNA. Once the RNA Polymerase reaches the termination site which is also known as the transcription stop site, mRNA transcript is complete and the DNA strand begins to rezip back into its original form which is the double helix. Page 1 of 9

3) Consider an enzyme that is composed of 3 different polypeptides. In addition, the enzyme is made up of duplicated of each polypeptide. In other words, the protein is made up of 6 subunits. What is the number of genes that code for this enzyme? 5 Points A) 2 B) 3 C) 4 D) 5 E) 6 4) The illustration below shows the structures of guanine (G) and cytosine (C), as they would be oriented in a DNA double helix. INDICATE the following: ALL POLARITY (delta + and delta -), and the hydrogen bonds (dashes) that stabilize this association. 10 Points Page 2 of 9

5) Let’s say the promoter region of a gene is determined by the sequence TTAGCGGGG. Find the two-promoter sequence in the gene below and indicate the direction the RNA polymerase would move for each . 10 Points 5’CACAGTTAGCGGGGTATCCGAG CCCCGCTAATT3’ 3’GTGTC AATCGCCCCATAGGCTCGGGGCGATTAA5’ 5’-3’: Left to Right promoter sequence -> CCCCGCTAA 3’-5’: Right to Left promoter sequence -> CCCCGCTAA 6). Label each of item indicated in the illustration below. 7 Points A________Amino Acid______________________ E__mRNA_______________________ B_______tRNA________________________ F_Ribosome large/ small subunit___________ C_____anticodons________________________ G_____5’ (five prime end) ______________ Page 3 of 9

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D_______3’ (three prime end) _______________ 7) The DNA sequence below corresponds to the gene for the enzyme hexokinase. Let’s say you want to amplify only the section of the gene that is in bold. 20 Points Design two 10 bp primers to do this. Clearly indicate the sequence and location of each primer and the direction the Taq polymerase would move TTCGCGGGAATGGGTGCGACCATGACAGTCTCTTTTTTCTGTATCGTGGAAATCATTTTCATTTTTATTG TTAGCTAATGCAATAGTTACTGAACTGATCCGATGAGTTAATGTTGAACAAATCTCATGTTGCGTGGTGG TCGCTTTTACCACAGATGCGTTTATGCCAGTATGGTTTGTTGAATTTTTATTAAATCTGGGTTGAGCGTG TCGGGAGCAAGTGTGAGCAGCAAAGTGGAACAACTGCGTGCGCAGTTAAATGAACGTATTCTGGTGCTGG ACGGCGGTATGGGCACCATGATCCAGAGTTATCGACTGAACGAAGCCGATTTTCGTGGTGAACGCTTTGC CGACTGGCCATGCGACCTCAAAGGCAACAACGACCTGCTGGTACTCAGTAAACCGGAAGTGATCGCCGCT ATCCACAACGCCTACTTTGAAGCGGGCGCGGATATCATCGAAACCAACACCTTCAACTCCACGACCATTG CGATGGCGGATTACCAGATGGAATCCCT 5’ GTCGGCGGAA 3’ ATCAACTTTGCGGCGGCGAAACTGGCGCGACG 3’ CAGCCGCCTT 5’ TTGTGCTGACGAGTGGACCGCGCGCACGCCAGAGAAACCGCGCTACGTTGCCGGTGTTCTCGGCCCGACC AACCGCACGGCGTCTATTTCTCCGGACGTCAACGATCCGGCATTTCGTAATATCACTTTTGACGGGCTGG TGGCGGCTTATCGAGAGTCCACCAAAGCGCTGGTGGAAGGTGGCGCGGATCTGATCCTGATTGAAACCGT TTTCGACACCCTTAACGCCAAAGCGGCGGTATTTGCGGTGAAAACGGAGTTTGAAGCGCTGGGCGTTGAG CTGCCGATTATGATCTCCGGCACCATCACCGACGCCTCCGGGCGCACGCTCTCCGGGCAGACCACCGAAG Page 4 of 9

CATTTTACAACTCATTGCGCCACGCCGAAGCTCTGACCTTTGGCCTGAACTGTGCGCTGGGGCCCGATGA ACTGCGCCAGTACGTGCAGGAGCTGTCACGGATTGCGGAATGCTACGTCACCGCGCACCCGAACGCCGGG CTACCCAACGCCTTTGGTGAGTACGATCTCGACGCCGACACGATGGCAAAACAGATACGTGAATGGGCGC AAGCGGGTTTTCTCAATATCGTCGGCGGCTGCTGTGGCACCACGCCACAACATATTGCAGCGATGAGTCG TGCAGTAGAAGGATTAGCGCCGCGCAAACTGCCGGAAATTC 5’ CCGTAGCCTG 3’ CCGTTTGTCCGGCCTGGAG 3’GGCATCGGAC 5’ CCGCTGAACATTGGCGAAGATAGCCTGTTTGTGAACGTGGGTGAACGCACCAACGTCACCGGTTCCGCTA AGTTCAAGCGCCTGATCAAAGAAGAGAAATACAGCGAGGCGCTGGATGTCGCGCGTCAACAGGTGGAAAA CGGCGCGCAGATTATCGATATCAACATGGATGAAGGGATGCTCGATGCCGAAGCGGCGATGGTGCGTTTT CTCAATCTGATTGCCGGTGAACCGGATATCGCTCGCGTGCCGATTATGATCGACTCCTCAAAATGGGACG 1 st 10bp primer: (original) GTCGGCGGAA  (designed strand) CAGCCGCCTT going in the direction left to right. 2 nd 10bp primer: (original)(3’-5’) CCGTAGCCTG  (designed strand)(5’-3’) GGCATCGGAC going in the direction right to left 8) Predict the amino acid sequence coded for by the mRNA sequence below. The ribosome binding site is UUAC. 10 Points 5’ CGCGCC UUACAAAUGUUUAUGUGAAAAAAAAA 3’ 9) The pGLO plasmid is shown below. How many nucleotides long is this plasmid? ____5371_______ 1 Point Is it single stranded or double stranded? ____double stranded___ 1 Point If the plasmid is cut with the restriction enzymes Pvu I and EcoRI, how many fragments will be generated? _________2__________ 1 Point Page 5 of 9 met Phe met Stop site Start site

Digestion of the pGLO plasmid (see previous page). Page 6 of 9 Let’s say you did a restriction endonuclease digestions of this plasmid. You used the enzymes PvuI, FspI, and EcoRI. List the sizes of the fragments below. 5 Points Pvu 1- 3054 EcoR1- 2063 Fsp 1- 3200 CLEARLY indicate the location of the fragments in lane 2 in the agarose gel below. 5 Points

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PVU1- 3054 ECOR1- 2063 FSP 1- 3200 10) Let’s say the image below shows an active site of an enzyme. The R -group structures are shown. Consider the following amino acid switches. Look at the amino acid R-groups when you think about this. A and D are both replaced by the amino acid leucine C is replaced by serine. Write the amino acid structures of leucine and serine below. Circle their R-groups. 5 Points Leucine: Serine: Page 7 of 9

Will these mutations stabilize the structure of the active site, or destabilize it? Explain. 10 Points I believe that the mutations of leucine and serine being replaced would cause the active site to destabilize. In proteins polarity play a role in the structure of the protein. So, by having leucine inside (which is a non- polar and hydrophobic), and serine (which is polar) it can destabilize the hydrogen bonds and the active site would be negatively affected. Page 8 of 9

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