Q 2010 by The International Union of Biochemistry and Molecular Biology BIOCHEMISTRY AND MOLECULAR BIOLOGY EDUCATION

Vol. 38, No. 4, pp. 268–269, 2010

Student Centered Education

Problem-based Test: An In Vitro Experiment to Analyze

the Genetic Code
Received for publication, May 10, 2010, and in revised from, May 20, 2010

József Szeberényi‡
From the Department of Medical Biology, Medical School, University of Pécs, H-7624 Pécs, Hungary

Terms to be familiar with before you start to solve the test: genetic code, translation, synthetic polynu-
cleotide, 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 A cytoplasmic extract was prepared from E. coli cells,

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 trans-
lated into the ‘‘text’’ of amino acid sequence in proteins.
incubated briefly at room temperature and then added to
the following three reaction mixtures:
1. mixture 1 contained leucine and a synthetic polyri-
bonucleotide (poly UC) consisting of U and C nucle-
otides in alternating sequence (þpoly UC þLeu);
2. mixture 2 contained poly UC without leucine
(þPoly UC –Leu);
3. mixture 3 contained leucine without poly UC (–Poly UC
þLeu).

[14C]Serine, ATP and GTP were also added to the

reaction tubes, the mixtures were incubated at 308C, and
aliquots were taken from them at the time points indi-
cated in Fig. 1, and treated with ice-cold trichloroacetic
acid (TCA)1 to precipitate macromolecules. The precipi-
tates were collected on filter discs, washed with TCA
and the radioactivity on the filters was determined in a
liquid scintillation counter. Study the experimental proto-
col and the results presented in Fig. 1 and solve the fol-
lowing multiple-choice questions (MCQs).

FIVE-CHOICE COMPLETION
(This type of question consists of a question or incom-
plete statement followed by five suggested answers or
FIG. 1. TCA-precipitated radioactivities in reaction mixtures completions. Select the one best answer.)
containing E. coli extracts, [14C]serine, ATP, GTP and/or poly
UC, leucine (l—lþPoly UC þLeu; *—* þPoly UC –Leu;
—
1. ____ What process was quantified by determining
–Poly UC þLeu; further details are described in the text).
TCA-insoluble radioactivites in this experiment?
A. Transcription of bacterial mRNA
B. Synthesis and processing of bacterial rRNA
‡ To whom correspondence should be addressed. Tel.: 36-72-
536216; Fax: 36-72-536453. E-mail: jozsef.szeberenyi@aok.pte.hu. C. Synthesis of polypeptides
1
The abbreviations used are: MCQ, multiple-choice question; D. A and B
TCA, trichloroacetic acid. E. A, B and C
DOI 10.1002/bmb.20421 268 This paper is available on line at http://www.bambed.org

2. ____ What was the aim of adding poly UC to some
of the mixtures?
A. To inhibit initiation of protein synthesis on en-
dogenous mRNA templates
B. To inhibit elongation of protein synthesis on en-
dogenous mRNA templates
C. A and B
D. To provide a template for protein synthesis
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. 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 contra-
dicted by the information given.)
4. ____ Aminoacyl-tRNA synthesis took place in the
reaction mixtures.
5. ____ Poly UC can serve as template in protein syn-
thesis.
6. ____ The polypeptides synthesized in the þPoly UC
þLeu mixture contain equal numbers of leucine and
serine.
7. ____ The E. coli extract contained ribosomes.
8. ____ In cell-free mixtures used in this experiment ini-
tiation of protein synthesis can take place on the ini-
tiation codon (AUG) only.
Five-choice Completion
(This type of question consists of a question or incom-
plete 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 pro-
ven that amino acids are determined by triplets of
nucleotides. In fact, at the time of this experiment
269
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: CUC codes for both serine and leucine
E: UCU or CUC code for leucine
CORRECT ANSWERS
1. C 6. A
2. D 7. A
3. C 8. B
4. A 9. E
5. A 10. E
EXPLANATIONS
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. trip-
lets) of the genetic code. Synthetic mRNAs of known
sequence were added to such mixtures after the degra-
dation of endogenous mRNA molecules (MCQ 2: D;
MCQ 3: C). Radioactively labeled amino acids are rapidly
linked to their cognate tRNAs by aminoacyl-tRNA syn-
thases 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 trip-
lets in poly UC, UCU, and CUC, follow each other with-
out 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).
REFERENCE
[1] H. G. Khorana (1977) Nobel Lectures in Molecular Biology 1933–
1975, Elsevier, New York, pp. 303–331.