Biochemical Genetics: DNA, RNA, and Protein Synthesis.

INTRODUCTION
Genetic information is coded for in GENES, which in turn are composed of a
compound called DEOXYRIBONUCLEIC ACID ("DNA" for short). Genes do
their work by telling a cell what type of proteins to make. Most of these
proteins are enzymes and they control attributes such as eye color, hair color,
and the ability to resist a particular disease (among other genetic TRAITS).
Eukaryotic organisms, like ourselves, have thread-like strands of DNA while
prokaryotes (bacteria, for example) have circular loops of DNA. Eukaryotic DNA
is also packaged (along with proteins) in cellular organelles called
CHROMOSOMES while prokaryotic DNA is not packaged in chromosomes.
Chromosomes are found in the nucleus and contain nearly all the DNA in a
eukaryotic cell.
Deoxyribonucleic acid belongs to a class of organic compounds called
NUCLEIC ACIDS. A related compound, RIBONUCLEIC ACID (RNA) is also
classified as a nucleic acid. Ribonucleic acid does its work outside the nucleus
in the cytoplasm. As we shall see, RNA is directly involved in protein synthesis.
____________________________________________________________________________

Simulating DNA Replication and RNA Transcription.

PURPOSE: To simulate replication and transcription using paper models.
MATERIALS NEEDED:
Scissors.
One picture of a DNA molecule with Bases.
Three dimensional models of DNA and RNA structure.
When a cell divides it must make an exact genetic duplicate of itself (except
for reproductive cells- eggs and sperm, or in abnormal cell division- cancers,
for example). If the copies are not exact then the DAUGHTER CELLS produced
by the division would not have the complete DNA instructions to synthesize
enzymes for metabolism. The parent cell must therefore copy its DNA before
reproducing and thus insure that each daughter cell receives complete genetic
information. DNA makes copies of itself in the nucleus of the cell; a process
called REPLICATION (Fig 1).

FIGURE 1. Replication of DNA

A second class of nucleic acids, the ribonucleic acids (RNA) are also
manufactured in the nucleus by a process known as TRANSCRIPTION.
Transcription
uses the instructions of DNA to synthesize RNA. There are three flavors of RNA
and, although they are synthesized in the nucleus, they do their work out in
the cytoplasm (Fig 2)

FIGURE 2 Comparison of Replication, Transcription, and Translation.

A third process (TRANSLATION) is the reason for all this replication and
transcription: protein synthesis. Translation uses RNA to bond together amino
acids to synthesize polypeptides. In brief, TRANSFER RNA (tRNA) picks up
amino acids and carries them to the site of protein synthesis at the ribosome
(Fig 2). The ribosomes are composed of protein and RIBOSOMAL RNA (rRNA).
There the instructions for making the protein are read from a strand of
MESSENGER RNA (mRNA). The transfer RNA then releases the amino acid and
is free to pick up another in the cytoplasm.
PROCEDURE:
1- Preparation. Work in pairs. Locate and remove one DNA template
sheet from one of your laboratory manuals. Separate the pieces by first
cutting along the green dashed lines. Each person works with one of the
base sheets while the DNA molecule is shared. Separate the bases by
cutting along the black dashed lines and then place them face-up on
the table. Keep your bases separate from those of your partner.
2- General DNA structure. Examine the crib sheet card with the
structural formulae and compare to the color drawing of the DNA
molecule, and three dimensional display model(s).
Your game pieces are color-coded as follows:

Adenine: Yellow-Brown.
Cytosine: Green.
Guanine: Blue.
Thymine: Red.

.
Deoxyribose: A black pentagon
Phosphate Group (PO 4): An blue circle

3- Deoxyribonucleic Acid Replication. The base pairing between purines

and pyrimidines allows DNA to serve as its own template to make accurate
copies of itself. To take advantage of the base pairing, DNA must unwind and
split lengthwise to expose the base pairs on each strand (Fig 1). Simulate the
initial separation of your DNA by cutting the colored DNA molecule along the
orange dashed line. Take one of the DNA chains

FIGURE 3. DNA Replication using the game pieces.

4- Single nucleotides are attracted to the proper exposed bases on the singlestranded DNA (Fig 3). Enzymes (the DNA POLYMERASES) are used to attach the
3' end of the deoxyribose to the previous phosphate. In this way, a new DNA
strand is built using the exposed strand as a template.
5- Use your nucleotide game pieces to build a new DNA molecule. Your strand
of DNA will serve as a template. Start at the 5' end of your DNA template (DNA
is always read from the 5' to the 3' end) and add nucleotides. Pair up thymine
(Red) with adenine (yellow) and cytosine (green) with guanine (blue; Fig 7).
Compare your replicated DNA molecule with that of your lab partner. They
should have identical sequences; otherwise one of you has
made a mistake
and have inadvertently simulated a mutation. Record your base sequences in
the results section.
6- Simulating a Mutation. In this exercise the colored DNA strand labeled for
person #1 (the one with the plain blue phosphates) will be used as a template
for replication. We will simulate our mutations at the sixth base from the 5' end
(thymine). Move in and pair up five nucleotide bases as before (read from the
5' end). At the sixth position insert a thymine instead of the correct base, and

then continue your replication as before. Note the "dimple" in your doublestranded DNA. This type of genetic damage is common when cells are exposed
to high levels ultraviolet radiation. Ultraviolet light is used in some bacterial
sterilizers and is responsible for causing sun burns and increasing the
incidence of skin cancer in humans (especially for those of you who enjoy
basking like lizards in the summer sun). The double thymine condition is called
a THYMINE DIMER. Other types of radiation or chemicals can also cause
changes in the DNA sequence.
7- Remove the long colored DNA strand and then use your mutated strand as a
template for further DNA replication. Compare your finished DNA molecule to
the original. What will happen when the mutated strand is replicated? This
simulation demonstrates how mutations can become fixed in the DNA. Record
your base sequences in the results section.
8- Transcription of Ribonucleic Acid. DNA serves not only as a template for
its own replication, but also provides instructions for RNA synthesis.
(Transcription; Fig 2). Ribonucleic Acid differs from DNA in several ways: While
the sugar molecule for DNA is deoxyribose, that for RNA is RIBOSE. Ribose
differs from deoxyribose in the substitution of a hydroxyl group (OH) for a
hydrogen (H; the change is shown in violet on the crib sheet). RNA is often
found in a single-stranded form while DNA is mostly doublestranded. RNA
also lacks thymine (the base URACIL is substituted). Uracil is
structurallyrelated to thymine (it is a single-ringed pyrimidine), but it has a hydrogen
instead of a methyl group (CH3) on one of the ring carbons. (see the crib
sheet).
9- Transcription follows more-or-less the same sequence of you simulated for
DNA replication: DNA molecule unwinds to expose a strand. The DNA template
of one strand (the TRANSCRIBED STRAND) is read in a 5' to 3' direction. The
other DNA strand (the NONCODING STRAND or NONTRANSCRIBED STRAND) is
not read. A class of enzymes called "RNA polymerases" bond
RIBONUCLEOTIDES (ribose attached to phosphates and a base) to one another
to form single-stranded RNA. The base-pairing is the same as that for DNA
except that adenine on the transcribed strand of the DNA attracts uracil
ribonucleotides rather than thymine.
10- Since RNA transcription is so similar to DNA replication, we won't bother
with a simulation. Answer the questions and exercises in the results section.
____________________________________________________________________________
Simulating Translation.
PURPOSE: To simulate translation and introduce the student to the genetic
code.
MATERIALS NEEDED:
Scissors.
Color sheet of mRNA, tRNA, and base code table
Amino acid/ribosome sheet
Now that you have seen how DNA and RNA are synthesized, we will
continue with a simulation of translation (protein synthesis).
PROCEDURE:
1- Preparation. Locate and remove the color sheet of messenger RNA
(mRNA), transfer RNA (tRNA), base code table, and full-color souvenir
bookmark. Also remove the amino acid/ribosome sheet. Cut along the dashed
lines and place the game pieces face-up. Cut the box out of the ribosome
and cut two slots in the paper as indicated.
2- Decoding the Message and Simulating Translation. Before translation
can be simulated you should first understand the relationship between the
mRNA codons and tRNA anticodons. You should also know the procedure used

to decode the message carried by the mRNA (Fig 4 and Base Code Table). The
abbreviations for the amino acids are listed in Table 1.
Amino Acid

Abbr.

Alanine
Arginine

ALA
ARG

Amino Acid
Glutamic
Acid
Glutamine

Abbr.

Amino Acid

Abbr.

GLU
GLN

Leucine
Lysine

LEU
LYS

Asparagine ASN
Glycine GLY
Aspartic
Acid ASP
Histadine HIS
Cysteine CYS
Isoleucine ILE
TABLE 1. Amino Acid Abbreviations.

Methionine
Phenylalanin
e
Proline

Amino
Acid

Abbr.
SER
THR

MET

Serine
Theonine
Tryptopha
n

PHE
PRO

Tyrosine
Valine

TYR
VAL

TRP

FIGURE 4. Relationship between codons, anticodons, and the genetic code.

3- Slide the long mRNA strand through the right-hand slot of the ribosome.
Position the start codon on the mRNA (AUG) at the left-hand side of the open
box on the ribosome (Fig 5A). This lines the start sequence (AUG) up with the
first groove on the ribosome. The first mRNA codon (AUG) is the "START"
command and corresponds to the amino acid methionine (Fig 4, Base Code
Table). The tRNA that interacts with a AUG codon is one with an anticodon
sequence of UAC (Fig 5A). In this diagram the UAC tRNA is shown with an
attached methionine. It is in position at the first groove of the ribosome and is
lined up with the start codon on the mRNA. At this point you have simulated
activation of the ribosome and protein synthesis can continue (activation is
actually more complex than this; see your text for details).

FIGURE 5. Reading the genetic code.

4- Determine the next amino acid required in the sequence by decoding the
next three bases. In our example the GCA codon corresponds to the amino acid
alanine (pay attention only to the text figures at this time, not the colored
game pieces). A tRNA with a complimentary CGU anticodon is picked from the
tRNA pool, and is used to transfer an alanine to the second groove (Fig 5A,B;
Fig 4). When the methionine and alanine are brought close to one another,
enzymes connect the two amino acids and release a molecule of water. A
hydrogen (H) and hydroxyl group (OH) are set off from your amino acid game
pieces by dotted lines to remind you of this. Note that, like DNA, the mRNA is
read from the 5' to the 3' end.
5- Following the formation of a bond between the two amino acids, the "older"
of the two tRNAs uncouples from its amino acid and leaves the ribosome (Fig
5). The newly-released tRNA can pick up another amino acid of the proper type
and be re-used. The ribosome slides toward the 3' end of the mRNA strand and
exposes the next codon on the mRNA and a third tRNA with an the proper
anticodon and attached amino acid is moved in (Fig 5A). This process
continues (Fig 5B) until the entire mRNA strand is read and a stop code is
encountered (UAA in our example). The polypeptide and mRNA are then
released and may be re-used (this depends on the organism)
6- Run through the translation simulation as described above. Use Figures 2, 4,
and 5 as guides. The color codes follow the same pattern as your DNA
replication simulation:
Adenine: Violet. Guanine: Blue. Cytosine: Green. Uracil: Red.

RNA anticodons must always match up with the codon on the mRNA. Adenine
(violet) pairs with Uracil (red) while the blue guanine and green cytosine form a
pair. The base code table is also color coded. Remember that the base code
table is keyed to the codon on the mRNA, not the anticodon on the rRNA.
Record the resulting sequence of amino acids in the results section and answer
the appropriate questions.

REPORT SECTION

by __________________________________
To complete your assignment for this exercise, fill in all the information
requested in the RESULTS section, tear out at the perforations and hand in.
This constitutes your Laboratory Report for this Experiment.
RESULTS
____________________________________________________________________________
Simulating DNA Replication and RNA Transcription.
Deoxyribonucleic Acid Replication. Record the sequence of bases for both
strands of your replicated DNA molecule and that of your partner.

Are the base sequences of the two DNA molecules equivalent to each other
and
the original? _______________________________________________________________
Simulating a Mutation. Record the sequences for both mutated DNA
molecules.

Are these sequences different from the original? ____________________________

What will all future DNA molecules replicated from the above pair look like
(assuming that there are no further mutations)?
_________________________________________________
_____________________________________________________________________________

Transcription of Ribonucleic Acid. Show that you understand the

relationship between DNA and RNA by filling in the blanks below the DNA
strand with a properly transcribed molecule of RNA (remember uracil):
3'
Single-stranded DNA to be transcribed
5'
T---A---C-----G---G---G-----T---T---T------C---A---T-------G---G---C----A---T---C
___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___
5' end
<-Codon 2-> <-Codon 3-> <-Codon 4-> <-Codon 5->
3' end
of RNA
of RNA
____________________________________________________________________________
Simulating Translation.
Decoding the Message and Simulating Translation.
Assume that the RNA you transcribed in the previous question is messenger
RNA.
Decode the codons for the above base sequence and indicate the resulting
polypeptide:
___________ ___________ ___________ ___________ ___________ ____________
We will call this peptide "ENZYME 1"
Consequences of Mutations at the Level of DNA. In this section you will
simulate the effects of a DNA mutation on translation. The underlined bases of
the DNA molecule in the previous section that transcribe codons two and three
of the above mRNA molecule will be affected.
Starting with the following mutated single strand of DNA, indicate the mRNA
that would be transcribed and the protein that would be translated (an adenine
has been substituted for the third base (guanine) that transcribes for mRNA
codon 2):
3'
Single-stranded DNA to be transcribed
5'
T---A----C----G---G---A----T----T---T----C---A----T----G---G---C-----AT---C
___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___
5' end
<-Codon 2-> <-Codon 3-> <-Codon 4-> <-Codon 5->
3' end
of mRNA
of
mRNA
Protein Translated from the Above mRNA
___________ ___________ ___________ ___________ ___________ ____________
We will call this peptide "ENZYME 2".
Does the protein made from the mutated strand (ENZYME 2) differ from the
unmutated protein (ENZYME 1)? Are there any base substitutions that can be
made at position three that will affect enzyme activity? Explain.
________________________________________
__________________________________________________________________________
__________________________________________________________________________
__________________________________________________________________________
__________________________________________________________________________
__________________________________________________________________________
Assume that the first base coding for the third codon (thymine) has been
exchanged for an adenine:
3'
Single-stranded DNA to be transcribed
5'
T----A---C----G---G---A----A----T---T-----C---A----T----G---G---C----A---T---C
___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___

5' end
of mRNA

<-Codon 2-> <-Codon 3-> <-Codon 4-> <-Codon 5->

3' end
of mRNA

Protein Translated from the Above mRNA

___________ ___________ ___________ ___________ ___________ ____________

Do you think this third enzyme will be functional (why or why not)?
_________________
__________________________________________________________________________
__________________________________________________________________________
__________________________________________________________________________
__________________________________________________________________________
__________________________________________________________________________
Working Backwards. A protein is sequenced and found to have the following
order of amino acids:
Methionine-Proline-Glycine-Cysteine-Alanine-Valine
Indicate a possible mRNA codon sequence that may have produced this
protein:
___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___
5' end
<-Codon 2-> <-Codon 3-> <-Codon 4-> <-Codon 5->
3' end
of mRNA
of mRNA

Indicate the base sequence of the DNA strand that transcribed your mRNA
strand:
___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___
3' end
5' end
of DNA
of DNA
And, just to finish things up, what does the other strand of your DNA look
like?
___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ___
5' end
3' end
of DNA
of DNA