NAME ________________________________

EXAM IV I. __________________/28November 25, 2002 Biochemistry I II. __________________/4

III. __________________/17

IV. __________________/8

V. __________________/6

VI. __________________/37

TOTAL /100

I. MULTIPLE CHOICE. (28 points; 2 points each)Choose the BEST answer to the question by circling the appropriate letter.

1. Which of the following is a palindromic sequence?

A. GGATCC B. GTATCC C. GAATCC D. AGGTCC E. CCTTCC

2. In double-stranded DNA:

A. the two strands are parallel.B. sequences rich in A-T base pairs are more difficult to denature than

those rich in G-C pairs.C. the structure is compatible only with a right-handed (never a

left-handed) helix.D. the two strands have complementary sequences.E. the sequence of bases has no effect on the overall structure.

3. Which of these statements about alternate forms of DNA is false?

A. B-form DNA predominates in aqueous solution; dehydration favors the A form.

B. Z-form DNA has a helical sense (left-handed) that is opposite that for A-form DNA.

C. The sugar pucker is either 2' or 3'.D. Nucleotide sequence has little or no effect on which form DNA takes.

4. Prokaryotic DNA polymerase III:

A. contains a 5' ® 3' proofreading activity to improve the fidelity of replication.

B. synthesizes DNA 3' ® 5'.C. has a b subunit that acts as a circular clamp to greatly improve the

processivity of DNA synthesis.D. synthesizes only the leading strand; DNA polymerase I synthesizes

the lagging strand.

5. Common lesions found in DNA after exposure to ultraviolet light are __.

A. pyrimidine dimersB. single strand breaksC. AP-sitesD. deamination of cytosineE. purine dimers

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6. Which of the following statements about aminoacyl-tRNA synthetases is false?

A. There is a different synthetase for every amino acid.B. The enzyme attaches an amino acid to the 3' end of a tRNA.C. The enzyme splits ATP to AMP + PP i .

D. The enzyme will use any tRNA species, but is highly specific for a given amino acid.

E. Some of the enzymes have an editing/proofreading capacity.

7. Which of the following statements about the polymerase chain reaction (PCR) is false?

A. DNA is amplified at random points when using genomic DNA as template.

B. The boundaries of the amplified DNA segment are determined by the synthetic oligonucleotides used to prime DNA synthesis.

C. The technique is useful for site-directed mutagenesis.D. Newly synthesized DNA must be denatured with heat before the next

round of DNA synthesis begins.E. DNA amplified by PCR can be cloned.

8. Which of the following is true about a circular double-stranded DNA genome that is determined by chemical means to be 21% adenosine?

A. The genome is 10.5% guanosine.B. The genome is 21% guanosine.C. The genome is 29% guanosine.D. The genome is 58% guanosine.E. The base percent composition of guanosine in the genome cannot be

determined from the information given.

9. Which of the following is most likely to lead to a loss of gene function?

A. A missense mutation (coding a different amino acid) in the open reading frame

B. A change from a TAA codon to a TAG codon (see page 7) in the protein coding region

C. A change from T to C in the promoter regionD. A frameshift mutation in the coding regionE. A sequence change in the 3'UTR

10. Which of the following is NOT involved in the processing of mRNA precursors in eukaryotic cells?

A. Capping of the 5'-endB. Addition of poly-AC. Excision of intronsD. Splicing of exonsE. Transport of the pre-mRNA to the cytoplasm

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11. The ribosome is involved in all of the following EXCEPT

A. peptide bond formationB. aminoacylation of tRNAC. binding of protein factors during elongationD. binding of aminoacyl tRNA to mRNAE. binding of mRNA at an initiation codon

12. Which of the following in NOT a potential problem associated with expressing a eukaryotic, protein-encoding gene in prokaryotic cells?

A. Lack of an intron-splicing mechanism in prokaryotesB. Differences in the translation initiation codon used by eukaryotic

cells and prokaryotic cellsC. Susceptibility of the protein product to prokaryotic proteasesD. Stability of mRNA in prokaryotic cellsE. Differences in transcriptional signals between eukaryotic cells and

prokaryotic cells

13. All of the following contribute to promoter binding by RNA polymerase in E. coli EXCEPT the

A. rho factorB. -10 consensus sequenceC. -35 consensus sequenceD. b' subunit of RNA polymeraseE. b subunit of RNA polymerase

14. In a bacterial cell, a mutation in an aminoacyl-tRNA synthetase leads to charging of the entire tRNA Ser population with alanine. Which of the following describes the result of using these aminoacyl tRNAs for protein synthesis in the cell?

A. The alanyl-tRNA Ser will not function in protein synthesis.B. Proteins synthesized using the alanyl-tRNA Ser will contain neither

alanine nor serine.C. Proteins synthesized using the alanyl-tRNA Ser will contain only

serine where alanine would normally occur.D. Proteins synthesized using the alanyl-tRNA Ser will contain only

alanine where serine would normally occur.E. Proteins synthesized using the alanyl-tRNA Ser will randomly contain

either alanine or serine where serine would normally occur.

II. BASE PAIRS. (4 points)

15. Draw the rare tautomeric form of guanosine. Place arrow(s) pointing to those hydrogen-bond donors or acceptors that might change due to the tautomeric shift.

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III. MATCHING (17 points)Follow directions to each problem.16. Match the type of bond with the role below:

Bond type Role (a) phosphodiester ___ links base to pentose in a nucleotide (b) N-glycosidic ___ joins adjacent nucleotides in one strand (c) phosphate ester ___ joins complementary nucleotides in two (d) hydrogen strands ___ difference between a nucleoside and a nucleotide.

17. Match the factor or enzyme at right with the stage(s) of protein synthesis at which it acts. If a factor or enzyme participates in two stages of protein synthesis, indicate all of them.

___ amino acid activation (a) RF1 ___ initiation (b) EF-Tu___ elongation (c) AA-tRNA synthetase___ synthetase termination (d) Shine-Dalgarno sequence

18. Match each feature of the plasmid pBR322 (at left) with one appropriate description presented (at right) (see picture of pBR322 below). Descriptions may be used more than once.

EcoRI

BamHI

SalI

( ori)

PvuII

PstI

( tet )R( amp )R

____ ampR sequence (a) permits selection of bacteria containing ____ ori sequence the plasmid ____ tetR (b) a sequence required for packaging ____ BamHI sequence recombinant plasmids into bacteriophage____ PstI sequence (c) origin of replication (d) cleavage of the plasmid here does not affect the antibiotic resistance genes (e) insertion of foreign DNA here permits selection of bacteria containing recombinant plasmids

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19. 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 an E & P if the statement applies to both eukaryotes and prokaryotes. ___ A single RNA polymerase transcribes genes that encode mRNAs, tRNAs, and rRNA. ___ RNA processing/stability is a level at which gene expression can be regulated. ___ Sigma ( s) subunit detaches from RNA polymerase shortly after transcription has initiated. ___ The 5' end of the mature mRNA begins with a triphosphate.

IV. Fill in the Blanks. (8 points)Write the appropriate word or words in each blank provided.

20. DNA replication in E. coli begins at a site in the DNA called the (a) ___________. At the replication fork the (b) ___________ strand is synthesized continuously while the (c) _________ strand is synthesized discontinuously. On the strand synthesized discontinuously, the short pieces are called (d) ____________ fragments. An RNA primer for each of the fragments is synthesized by an enzyme called (e) __________, and this RNA primer is removed after the fragment is synthesized by the enzyme (f) ___________, using its (g) _____________ activity. The nicks left behind in this process are sealed by the enzyme (h) _____________.

V. TRUE/FALSE. (6 points)Choose the BEST answer to the question by circling the appropriate letter.

T F 21. The A-form and B-form DNA are right-handed helices, but Z-form DNA is a left-handed helix found only in single-stranded DNA.

T F 22. The linking number ( Lk) of DNA can be changed only by breaking one or both strands of constrained DNA.

T F 23. All tRNAs have the sequence -CCA at their 5' terminus.

T F 24. Some tRNAs contain G=U base pairs.

T F 25. Ribosomes contain many separate proteins.

T F 26. The three ribosomal RNAs in a bacterial ribosome are distributed in three separate large ribosomal subunits.

VI. SHORT ANSWER (37 points)Give a very brief answer as directed to each problem or question below.

27. What is the approximate length of a DNA molecule in mmeters (in the B form) containing 10,000 base pairs? (2 pts)

28. Describe the composition and structure of a nucleosome. (3 pts)

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29. Why does lowering the ionic strength of a solution of double-stranded DNA permit the DNA to denature more readily (for example, to denature at a lower temperature than at a higher ionic strength)? (2 pts)

30. Transfer RNAs have several bases in addition to the normal four found in RNA. How are these rare bases incorporated into the tRNA molecule? (2 pts)

31. Below is a piece of DNA that is giving rise to an RNA molecule. Indicate the 5' and 3' ends of the RNA molecule and of the strand of DNA that is complementary to the RNA molecule. In which direction is synthesis occurring (i.e., which end will be getting longer)? (4 pts)

32. List one basic property that distinguishes RNA polymerases from DNA polymerases, and list one basic property they share. (4 pts)

33. A new antibiotic named ericamycin was recently discovered by a brilliant graduate student. This antibiotic works by inhibiting prokaryotic protein synthesis. In the presence of ericamycin protein synthesis can initiate, but only dipeptides that remain bound to the ribosome are formed. What specific step of protein synthesis is likely to be blocked by this antibiotic? (3 pts)

34. In a certain organism, the gene for hexokinase has 20,000 base pairs. The molecular weight of hexokinase is 100,000. Is the organism likely to be a eukaryote or a prokaryote? Explain your answer (do any calculations on the back). (2 pts)

35. In protein synthesis, 61 codons specify the 20 amino acids. Base pairing between the codon and the tRNA anticodon assures that the correct amino acid will be inserted into the nascent polypeptide chain. Why then does the cell require only 32 different tRNAs to recognize 61 different codons? (3 pts)

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The Standard Genetic Code

UUU Phe UCU Ser UAU Tyr UGU Cys

UUC Phe UCC Ser UAC Tyr UGC Cys

UUA Leu UCA Ser UAA Stop UGA Stop

UUG Leu UCG Ser UAG Stop UGG Trp

CUU Leu CCU Pro CAU His CGU Arg

CUC Leu CCC Pro CAC His CGC Arg

CUA Leu CCA Pro CAA Gln CGA Arg

CUG Leu CCG Pro CAG Gln CGG Arg

AUU Ile ACU Thr AAU Asn AGU Ser

AUC Ile ACC Thr AAC Asn AGC Ser

AUA Ile ACA Thr AAA Lys AGA Arg

AUG Met ACG Thr AAG Lys AGG Arg

GUU Val GCU Ala GAU Asp GGU Gly

GUC Val GCC Ala GAC Asp GGC Gly

GUA Val GCA Ala GAA Glu GGA Gly

GUG Val GCG Ala GAG Glu GGG Gly

36. The template strand of a segment of double-stranded DNA contains the sequence:

(5')CTT TGA TAA GGA TAG CCC TTC

(a) What is the base sequence of the mRNA that can be transcribed from this strand (list in 5' ® 3' direction)? (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? (Refer to the genetic code table.) (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? (6 pts)

37. What is the basis of the separation on your cellulose acetate strips in the laboratory? Draw the expected pattern of bands you would expect from a zymogram run at pH 8.8 loaded with an enzyme composed of two isozymes ( a & b). Each isozyme has a trimeric quaternary structure and can associate with itself and each other. One isozyme has a net charge of +3/subunit and the other has a net charge of 0 at pH 8.8. Be sure to label the origin (or point of loading) and the anode and the cathode. (6 pts)

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Answer Sheet for Exam 4 11/25/2002

Test CorrectQuestion Answer

Multiple Choice1 A2 D3 D4 C5 A6 D7 A8 C9 D

10 E11 B12 B13 A14 D

Base Pairs15

N

NN

N

7

8

9

1

2

3

4

5

6

H N

H

OH

N

NN

N

7

8

9

1

2

3

4

5

6

H N

H

O

H

Enol

Keto

O2HO-CH

O2HO-CH

ribose

ribose

----Matching

16 b; a; d; c17 c; d; b; a18 a; c; a; e; e 19 P; E & P; P; P

Fill in the Blanks20 (a) origin; (b) leading; (c) lagging; (d) Okazaki;

(e) primase; (f) DNA pol I; (g) 5' ® 3' exonuclease;

(h) DNA ligase

Test CorrectQuestion Answer

True/False21 F22 T23 T24 T25 T26 F

Short Answer27 10,000 bp @10 bp/turn = 1000 turns @34 Å/turn = 34,000

Å

= 3,400 nm = 3.4 mm28 A nucleosome consists of double-stranded DNA wound

around a core of 8 histones. The octomer consists of 2 each of histone H2A, H2B, H3 and H4. Each nucleosome involves about 150 base pairs of DNA, and nucleosomes are spaced along a DNA molecule at intervals of about 200 base pairs.

29 Lower ionic strength reduces the screening of the negative charges on the phosphate groups by positive ions in the medium. The result is stronger charge- charge repulsion between the phosphate, which favors strand separation.

30 The unusual bases in tRNA are added by enzymatically modifying specific nucleotide residues in the pre-tRNA molecule.

31

3'

5'

3'

5'

direction ofsynthesis

32 Among the distinguishing characteristics: RNA polymerase does not require a primer, but DNA polymerase does; RNA polymerase lacks the 3' ®5' proofreading exonuclease activity present in DNA polymerase. Among the shared properties: both enzymes use nucleoside triphosphates as substrates, require Mg 2+, Zn 2+, produce an antiparallel complement to the template, and synthesize nucleic acids in the direction 5'® 3'.

33 Ericamycin probably blocks translocation. 34 The organism is a eukaryote.

-20,000 base pairs can encode a sequence of up to about 6700 amino acids. An enzyme of M

r 100,000 has about 900

amino acids (each amino acid residue contributes an average of 110 to the total molecular weight). The gene is therefore about seven times longer than needed to encode the enzyme; six-sevenths of the gene sequence must be introns, which are very rare in prokaryotes, but common in eukaryotes.

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Test CorrectQuestion Answer

35In each recognized codon, there is a standard anticodon- codon base pair with the first two bases of the codon; in the third base pair a "wobble base" exists that allows one tRNA to read three different codons. Similarly, tRNAs with U or G in the first anticodon position also exhibit a wobble effect that permits pairing with two different codons. For example, certain tRNAs have the unusual nucleotide inosinate in the first anticodon position. Because inosinate can base pair with A, U, or C, a tRNA containing hypoxanthine can recognize three different codons.

36(a) (5')GAA GGG CUA UCC UUA UCA AAG(b) Glu-Gly-Leu-Ser-Leu-Ser-Lys(c) The codons translate to Leu-Stop-Stop. No peptide would be produced, because of the stop codons.

37The basis of the separation is by the charge on the protein trimer. One trimer would have a net charge of +9, another +6, another +3, and the last (the one that does not move off the origin) is 0.

cathodeanode

origin

a b2a3 b3ab2

----

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