BIOL0280 Third Midterm Examination 2013

Biological amines are generated by decarboxylation of amino acids . 2. [2 points] The hormones epinephrine and norepinephrine are derived biosynthetically from: A) arginine. B) histidine. C) isoleucine. D) tryptophan. E) tyrosine. Circle the correct answer 3. [4 points] Draw the structure of UMP in the space provided below. Your answer must be completely correct to receive credit. 4. [2 points] Ribonucleotide reductase is a key enzyme in nucleotide biosynthesis. In one sentence, describe its biochemical function. Ribonucleotide reductase converts ribonucleotides to deoxyribonucleotides.

Explain the function of the two regulatory sites of ribonucleotide reductase, The primary regulatory site controls overall enzymatic activity and the substrate specificity site controls relative enzymatic activity i.e., which substrates are preferentially reduced. and list molecules that bind to each of these two regulatory sites. Primary site: ATP and dATP Specificity site: ATP, dATP, dGTP, dTTP 6. [2 points] The difference between a ribonucleotide and a deoxyribonucleotide is: A) a deoxyribonucleotide has an —H instead of an —OH at C-2. B) a deoxyribonucleotide has α configuration at C-1; a ribonucleotide has β configuration. C) a ribonucleotide has an extra —OH at C-4. D) a ribonucleotide has more structural flexibility than deoxyribonucleotide. E) a ribonucleotide is a pyranose, deoxyribonucleotide is a furanose. Circle the correct answer 7. [2 points] Which of the following are possible base compositions for double-stranded DNA? %A %G %C %T %U A) 5 45 45 5 0 B) 20 20 20 20 20 C) 35 15 35 15 0 D) All of the above. E) None of the above. Circle the correct answer.

In the Watson-Crick model of DNA structure (now called B-form DNA): A) a purine in one strand always hydrogen bonds with a purine in the other strand. B) A–T pairs share three hydrogen bonds. C) G–C pairs share two hydrogen bonds. D) the 5 ‘ ends of both strands are at one end of the helix. E) the bases occupy the interior of the helix. Circle the correct answer. 9. [2 points] Nucleosomes: A) are important features of chromosome organization in bacteria. B) contain proteins that are rich in acidic amino acids. C) are composed of protein and RNA. D) bind DNA in the form of a solenoidal supercoil. E) occur in chromatin at irregular intervals along the DNA molecule. Circle the correct answer. 10. [6 points] Calculate values for the following topological properties of a closed-circular DNA molecule containing 2,000 base pairs (for simplicity, assume there are 10 base pairs per turn in the relaxed DNA). (a) What is the linking number (Lk) when the DNA is 200 relaxed? (b) What is the linking number (Lk) when the DNA has 198 been underwound by 2 turns? (c) Will the DNA in (b) adopt positive or negative negative supercoils? 11. [2 points] When a DNA molecule is described as replicating bidirectionally, that means that it has two: A) chains. B) independently replicating segment. C) origins. D) replication forks. E) termination points. Circle the correct answer.

Briefly describe the biochemical role of the following enzymes in DNA replication in E. coli: (a) DNA helicase unwinds double-stranded DNA during replication (b) Primase synthesizes RNA primers (c) the 3 ‘ → 5′ exonuclease activity of DNA polymerase proofreads newly synthesized DNA by removing mismatched nucleotides (d) the 5′ → 3’ exonuclease activity of DNA polymerase I removes RNA primers 13. [6 points] In the space below, draw the product of the deamination reaction for the base shown on the left: deamination Name the bases: Cytosine Uracil Which repair pathway is most likely to repair this lesion? The base excision repair pathway

Below, an RNA molecule is being transcribed from a strand of DNA. (a) Label the 5′ and 3′ ends of the RNA molecule. (b) Label the 5′ and 3′ ends of the DNA template strand. (c) Using an arrow, indicate the direction of synthesis. 3´ 5´ 3´ 5´ 15. [5 points] For each of the following statements, indicate with a P if the statement applies only to prokaryotes, an E if the statement applies only to eukaryotes, and a B if the statement applies to both eukaryotes and prokaryotes. _B_ RNA polymerase initiates transcription de novo, with no requirement for a primer. _ E_ Promotor sequences have little intrinsic affinity for RNA polymerase. _P_ A single RNA polymerase transcribes genes that encode mRNAs, tRNAs, and rRNA. _P_ Sigma (σ) subunit detaches from RNA polymerase after transcription has initiated. _ E_ At the 5′ end of the mature mRNA there is a cap consisting of a guanosine joined to the 5′-terminal nucleotide through a 5 ‘ to 5 ‘ triphosphate group. 16. [2 points] A branched (“lariat”) structure is formed during: A) attachment of a 5’ cap to mRNA. B) attachment of poly(A) tails to mRNA. C) processing of preribosomal RNA. D) splicing of all classes of introns. E) splicing of group II introns. Circle the correct answer. 17. [2 points] Splicing of introns in nuclear mRNA primary transcripts requires: A) a guanine nucleoside or nucleotide. B) endoribonucleases. C) polynucleotide phosphorylase. D) RNA polymerase II. E) small nuclear RNAs (snRNAs). Circle the correct answer.

The template strand of a segment of double-stranded DNA contains the sequence: (5ʹ)CTT TGA TAA GGA TAG CCC TTC(3ʹ) (a) What is the base sequence of the mRNA that can be transcribed from this strand? Be certain to label all 5 ʹ and 3 ʹ ends in your answer. (5ʹ)GAA GGG CUA UCC UUA UCA AAG(3ʹ) (b) What amino acid sequence could be coded by the mRNA base sequence in (a), using only the first reading frame starting at the 5 ‘ end? (You may refer to the genetic code on the last page of the exam.) Glu-Gly-Leu-Ser-Leu-Ser-Lys (c) Suppose the other (complementary) strand is used as a template for transcription. What is the amino acid sequence of the resulting peptide, again starting from the 5’ end and using only the first reading frame? The codons translate to Leu-Stop-Stop. No peptide would be produced because of the stop codons. 19. [7 points] Match the factor or enzyme at the left with the stage(s) of protein synthesis at which it acts. If a factor or enzyme participates in two stages of protein synthesis, indicate both of them. A) RF C Amino acid activation B) EF-G D, E Initiation C) aminoacyl-tRNA B Elongation D) Shine-Dalgarno sequence E) IF-3 A, B, E Termination 20. [2 points] Approximately how many high-energy phosphate bonds must be consumed to incorporate one amino acid into a protein? (I.e., per peptide bond.) A) 0 B) 1 C) 2 D) 4 E) 8 Circle the correct answer.

In one or two sentences, define the following terms with respect to regulation of transcription: (a) Constitutive Refers to constant, unregulated transcription (b) Induction Refers to increased transcription through activation or positive regulation (c) Repression Refers to decreased transcription through negative regulation (d) Repressor Acts to impede access of RNA polymerase to promoters. (e) Co-activator Mediates interactions between RNA polymerase and transactivators. Does not bind DNA directly. 22. [4 points] In prokaryotes such as E. coli, many operons that encode enzymes involved in amino acid biosynthesis begin with a sequence coding for a leader peptide. This peptide has no known enzymatic function and is rich in the amino acid that is synthesized by the enzymes coded for in the operon. What is the function of this leader peptide? The leader peptide is integral to the regulatory mechanism called transcription attenuation. In prokaryotes, transcription and translation are closely coupled. The ability to translate the leader peptide signals that the relevant amino acid is readily available in the cell and that transcription of the operon is undesirable at this time. Translation through the leader peptide (when the amino acid is abundant) allows the formation of a hairpin attenuator structure downstream in the nascent RNA chain. This attenuator acts as a transcription terminator, causing dissociation of the RNA polymerase. When the relevant amino acid is in short supply, the ribosome stalls trying to synthesize the leader peptide. A different secondary structure forms in the nascent RNA, preventing formation of the terminator and allowing continued transcription of the operon.

Certain restriction enzymes produce cohesive (sticky) ends. This means that they: A) cut both DNA strands at the same base pair. B) cut in regions of high GC content, leaving ends that can form more hydrogen bonds than ends of high AT content. C) make a staggered double-strand cut, leaving ends with a few nucleotides of single- stranded DNA protruding. D) make ends that can anneal to cohesive ends generated by any other restriction enzyme. E) stick tightly to the ends of the DNA they have cut. Circle the correct answer. 24. [3 points] Match each vector with the appropriate method used for its introduction into host cells: (a) Electroporation b Plasmid (b) Transformation c Bacteriophage λ (c) Infection a BAC (Bacterial Artificial Chromosome) 25. [3 points] In order to amplify Gene X shown below using PCR, to what region and strand would the two oligonucleotide primers anneal? A) Region A and B of the top strand. B) Region A of both strands. C) Region A of the top strand and region B of the bottom strand. D) Region A of the bottom strand and region B of the top strand. E) None of the above. Circle the correct answer and draw the primers on the diagram.

A plasmid that encodes resistance to ampicillin and tetracycline is digested with the restriction endonuclease PstI, which cuts the plasmid at a single site in the ampicillin- resistance gene. The cut plasmid DNA is then ligated with a PstI digest of human DNA and introduced into E. coli cells. (a) What antibiotic would you put in an agar plate to ensure that the cells of a bacterial colony contain the plasmid? tetracycline (b) What will the antibiotic-resistance phenotypes be of the bacteria growing on the plate? R S (In your answer, use tet to refer to tetracycline resistance, tet to refer to tetracycline R S sensitivity, amp to refer to ampicillin resistance and amp to refer to ampicillin sensitivity.) R R tet amp R S tet amp (c) Which phenotype will indicate the presence of plasmids that contain human DNA fragments? Provide a brief explanation. R S tet amp This phenotype indicates that the gene for ampicillin resistance has been interrupted by the insertion of a human DNA fragment.

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