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Abstract

  1. Top of page
  2. Abstract
  3. THE EXPERIMENT
  4. FIVE-CHOICE COMPLETION
  5. CORRECT ANSWERS
  6. EXPLANATIONS
  7. REFERENCE

Terms to be familiar with before you start to solve the test: genetic code, translation, synthetic polynucleotide, leucine, serine, filter precipitation, radioactivity measurement, template, mRNA, tRNA, rRNA, aminoacyl-tRNA synthesis, ribosomes, degeneration of the code, wobble, initiation, and elongation of protein synthesis, initiation codon


THE EXPERIMENT

  1. Top of page
  2. Abstract
  3. THE EXPERIMENT
  4. FIVE-CHOICE COMPLETION
  5. CORRECT ANSWERS
  6. EXPLANATIONS
  7. REFERENCE

In the following problem-solving test a simple, but very important experiment carried out in the laboratory of the Nobel laurate Har Gobind Khorana is described [1]. The experiment belongs to a series of studies designed in the early years of molecular biology to decipher the genetic code: the mechanism by which the “text” written in the sequence of nucleotides of nucleic acids is translated into the “text” of amino acid sequence in proteins.

A cytoplasmic extract was prepared from E. coli cells, incubated briefly at room temperature and then added to the following three reaction mixtures:

  • 1
    mixture 1 contained leucine and a synthetic polyribonucleotide (poly UC) consisting of U and C nucleotides in alternating sequence (+poly UC +Leu);
  • 2
    mixture 2 contained poly UC without leucine (+Poly UC –Leu);
  • 3
    mixture 3 contained leucine without poly UC (–Poly UC +Leu).

[14C]Serine, ATP and GTP were also added to the reaction tubes, the mixtures were incubated at 30°C, and aliquots were taken from them at the time points indicated in Fig. 1, and treated with ice-cold trichloroacetic acid (TCA)1 to precipitate macromolecules. The precipitates were collected on filter discs, washed with TCA and the radioactivity on the filters was determined in a liquid scintillation counter. Study the experimental protocol and the results presented in Fig. 1 and solve the following multiple-choice questions (MCQs).

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Figure 1. TCA-precipitated radioactivities in reaction mixtures containing E. coli extracts, [14C]serine, ATP, GTP and/or poly UC, leucine (•—•+Poly UC +Leu; ○—○ +Poly UC –Leu; ⊙—⊙ –Poly UC +Leu; further details are described in the text).

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FIVE-CHOICE COMPLETION

  1. Top of page
  2. Abstract
  3. THE EXPERIMENT
  4. FIVE-CHOICE COMPLETION
  5. CORRECT ANSWERS
  6. EXPLANATIONS
  7. REFERENCE

(This type of question consists of a question or incomplete statement followed by five suggested answers or completions. Select the one best answer.)

  • 1.
    ____ What process was quantified by determining TCA-insoluble radioactivites in this experiment?
    • A.
      Transcription of bacterial mRNA
    • B.
      Synthesis and processing of bacterial rRNA
    • C.
      Synthesis of polypeptides
    • D.
      A and B
    • E.
      E.A, B and C
  • 2.
    ____ What was the aim of adding poly UC to some of the mixtures?
    • A.
      To inhibit initiation of protein synthesis on endogenous mRNA templates
    • B.
      To inhibit elongation of protein synthesis on endogenous mRNA templates
    • C.
      A and B
    • D.
      To provide a template for protein synthesis
    • E.
      E.None of the above is true
  • 3.
    ____ What was the aim of incubating the bacterial extract at room temperature, before setting up the mixtures?
    • A.
      A.To degrade chromosomal DNA
    • B.
      To degrade plasmid DNA
    • C.
      To degrade cellular mRNAs
    • D.
      To degrade cellular tRNAs
    • E.
      To degrade all cellular nucleic acids

Experiment Analysis

(The following statements are related to the information presented in the description of the experiment. Based on the information given, select:

  • A.
    if the statement is supported by the information given;
  • B.
    if the statement is contradicted by the information given;
  • C.
    if the statement is neither supported nor contradicted by the information given.)
  • 4.
    ____ Aminoacyl-tRNA synthesis took place in the reaction mixtures.
  • 5.
    ____ Poly UC can serve as template in protein synthesis.
  • 6.
    ____ The polypeptides synthesized in the +Poly UC +Leu mixture contain equal numbers of leucine and serine.
  • 7.
    ____ The E. coli extract contained ribosomes.
  • 8.
    ____ In cell-free mixtures used in this experiment initiation of protein synthesis can take place on the initiation codon (AUG) only.

Five-choice Completion

(This type of question consists of a question or incomplete statement followed by five suggested answers or completions. Select the one best answer.)

  • 9.
    ____ Which of the following features of the genetic code was supported by this experiment? (To be able to answer this question we should assume proven that amino acids are determined by triplets of nucleotides. In fact, at the time of this experiment the triplet nature of the genetic code was already known.)
    • A.
      The genetic code is degenerate
    • B.
      The genetic code is characterized by wobble
    • C.
      The genetic code is commaless
    • D.
      The genetic code has no overlaps
    • E.
      C and D
  • 10.
    ____ What can be the final conclusion of the experiment?
    • A:
      UCU codes for serine, CUC codes for leucine
    • B:
      UCU codes for leucine, CUC codes for serine
    • C:
      UCU codes for both serine and leucine
    • D:
      D: CUC codes for both serine and leucine
    • E:
      UCU or CUC code for leucine

CORRECT ANSWERS

  1. Top of page
  2. Abstract
  3. THE EXPERIMENT
  4. FIVE-CHOICE COMPLETION
  5. CORRECT ANSWERS
  6. EXPLANATIONS
  7. REFERENCE
Table  . 
1. C6. A
2. D7. A
3. C8. B
4. A9. E
5. A10. E

EXPLANATIONS

  1. Top of page
  2. Abstract
  3. THE EXPERIMENT
  4. FIVE-CHOICE COMPLETION
  5. CORRECT ANSWERS
  6. EXPLANATIONS
  7. REFERENCE

TCA filter precipitation of in vitro protein synthesizing mixtures (MCQ 1: C) containing all the components required for translation (mRNA template, tRNAs to carry the amino acids, ribosomes, ATP for aminoacyl-tRNA synthesis, GTP for ribosome translocation, amino acids) was an important technique to determine words (i.e. triplets) of the genetic code. Synthetic mRNAs of known sequence were added to such mixtures after the degradation of endogenous mRNA molecules (MCQ 2: D; MCQ 3: C). Radioactively labeled amino acids are rapidly linked to their cognate tRNAs by aminoacyl-tRNA synthases present in the E. coli cell extract (MCQ 4: A), and then incorporated into newly synthesized polypeptide chains (MCQ 5: A; MCQ 7: A; MCQ 8: B). The fact that incorporation of [14C]serine absolutely depends on the presence of leucine indicates that the two possible triplets in poly UC, UCU, and CUC, follow each other without overlapping of the triplets or leaving out nucleotides (“commas”) leading to the synthesis of polypeptides with alternating serines and leucines in them (MCQ 6: A; MCQ 9: E). That UCU codes for serine and CUC codes for leucine was determined by other experiments (MCQ 10: E).

  • 1

    The abbreviations used are: MCQ, multiple-choice question; TCA, trichloroacetic acid

REFERENCE

  1. Top of page
  2. Abstract
  3. THE EXPERIMENT
  4. FIVE-CHOICE COMPLETION
  5. CORRECT ANSWERS
  6. EXPLANATIONS
  7. REFERENCE
  • 1
    H. G. Khorana ( 1977) Nobel Lectures in Molecular Biology 1933–1975, Elsevier, New York, pp. 303331.