Freeman, Biological Science, 4e, Chapter 16 Instructor Guide 1

Chapter 16 - Transcription, RNA Processing, and Translation

Learning Objectives: Students should be able to
Relate the structure of RNA polymerase to its function in transcription.
Describe the steps in initiation, elongation, and termination of
transcription and translation.
Explain how RNA is processed in eukaryotes.
Relate the structure of ribosomes and tRNA to their functions in
translation.
Lecture Outline
Proteins are the stuff of life.
The role of proteins
Proteins control most cell processes like chemical reactions and also
give cells their structure and shape.
Cell function is dependent on protein function.
If one or more proteins do not work correctly, the cell may become
abnormal and die.
The central dogma
Proteins are synthesized in a two-step process: transcription of genes
into messenger RNAs and translation of messenger mRNAs into
proteins.
I. An Overview of Transcription
A. RNA polymerase enzyme
1. This enzyme is responsible for synthesizing RNA from DNA.
2. The enzyme uses only one of the DNA strands as a template.
a. This is called the template strand.
b. The other strand is called the non-template or coding strand.
c. The coding strand matches the mRNA sequence, except that it
has thymine where the mRNA has uracil.
B. Characteristics of RNA polymerase
1. RNA polymerase reads the DNA template and synthesizes a
complementary RNA strand. (Fig. 16.1)
2. It builds RNA in the 5' 3' direction by reading the DNA template.
3. It does not require a primer to begin RNA synthesis.
4. Bacteria have one RNA polymerase; eukaryotes have three distinct
types: pol I, pol II, and pol III. (Table 16.1)
a. RNA polymerase I transcribes genes that code for ribosomal
RNAs.
b. RNA polymerase II transcribes genes that code for proteins; thus,
it synthesizes mRNAs.
c. RNA polymerase III transcribes genes that code for tRNAs and
other small RNAs.
C. Initiation: How does transcription begin?
1. In bacteria, a protein, called sigma factor, binds to RNA polymerase
before transcription begins.

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Freeman, Biological Science, 4e, Chapter 16 Instructor Guide 2

a. RNA polymerase + sigma = holoenzyme.

b. RNA polymerase is the core enzyme. (Fig. 16.2a)
2. David Pribnow studied promoters.
a. Sigma binds tightly to specific regions of DNA called promoters.
b. Pribnow analyzed the base sequence of promoters
(1) Promoters are 4050 base pairs long.
(2) They have a series of bases similar or identical to TATAAT,
known as the 10 box
(3) The 10 box is 10 base pairs upstream from where
transcription will start (the +1 site).
(4) Another conserved sequence, TTGACA, occurs in the same
promoters about 35 bases upstream of the start site; it is
called the 35 box.
(5) In eukaryotic cells, most promoters include a unique
sequence called the TATA box, centered about 30 base pairs
upstream from the transcription initiation site.
c. Researchers found that sigma is required to facilitate RNA
polymerase binding to DNA.
d. The holoenzyme binds to DNA at specific locations called
promoters.
e. Promoters are sites on the DNA template where transcription
begins.
f. Sigma appeared to be responsible for guiding RNA polymerase
to promoters.
3. The role of sigma subunits and basal transcription factors
a. Transcription begins when sigma binds to the 35 and the 10
boxes. (Fig. 16.2b)
b. Bacteria have several different types of sigma proteins that each
bind to different promoters, determining which genes are
transcribed at a given time.
c. Eukaryotic basal transcription factors
(1) Analogous to sigma proteins in prokaryotes.
(2) Involve many proteins
(3) Not part of a holoenzyme
(4) Bind to the appropriate region of DNA
(5) Followed by RNA polymerase binding
4. Events inside the holoenzyme
a. After sigma binds to a promoter for a bacterial gene, the DNA
double helix opens.
b. The template strand is threaded through a channel in RNA
polymerase that leads to the active site.
c. Ribonucleoside triphosphates (NTPs) enter another channel in
RNA polymerase, move to the active site, and are incorporated
into the mRNA when they are complementary to the template
strand.
d. Sigma is released once RNA synthesis is under way.

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Freeman, Biological Science, 4e, Chapter 16 Instructor Guide 3

D. Elongation and termination

1. Elongation
a. RNA polymerase moves in the 3' 5' direction along the
template DNA strand
b. In the interior of the enzyme, a group of amino acids called the
rudder helps steer the template and non-template strands
through channels inside enzyme. (Fig. 16.3, step 3)
c. RNA polymerase catalyzes the addition of the nucleotide to the
3' end of the growing RNA molecule at a rate of about 50
nucleotides per second.
d. It inserts a uracil whenever it encounters adenine in the
template DNA.
e. A group of amino acids called the enzymes zipper helps
separate the new strand from the template strand.
f. An enzymes structure is correlated with its function.
(1) Double-stranded DNA goes into and out of one groove.
(2) NTPs enter another channel.
(3) The growing RNA structure exits through the rear.
2. Termination
a. Specific sequences in template DNA act as termination sites.
(1) In bacteria, the bases in the transcriptional termination
sequence form complementary base pairs.
(2) This results in a hairpin structure, which causes the RNA
strand to separate from the RNA polymerase, terminating
transcription. (Fig. 16.4)
b. RNA polymerase and the mRNA strand are released from the
DNA template.
c. Specific termination proteins facilitate the release in some
bacteria.
3. Students should be able to draw the RNA polymerase
holoenzyme; label sigma and the RNA polymerase core
enzyme and describe their function; and diagram the
initiation, elongation, and termination phases of transcription.
II. RNA Processing in Eukaryotes
A. The startling discovery of eukaryotic genes in pieces
1. In prokaryotes, when transcription of mRNA is complete, the
mRNA is ready to be converted into a protein.
2. In eukaryotes, pre-mRNA is produced by transcription, and this
must be processed before translation can occur.
3. Eukaryotic genes have intervening sequences called introns.
a. The protein-coding region of eukaryotic genes is interrupted by
stretches of noncoding DNA.
b. Noncoding sequences must be disposed of to make a functional
mRNA.
c. Eukaryotic gene organization is very different from that in
prokaryotes.

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Freeman, Biological Science, 4e, Chapter 16 Instructor Guide 4

4. P. Sharp and colleagues detected noncoding regions in genes.

a. They mixed together mRNA and DNA.
b. Then they incubated the mixture under conditions that promote
hybridization of the mRNA to its DNA template.
c. Following hybridization, they used electron microscopy to
observe the molecules in the solution.
d. Result: All of the mRNA was paired to DNA, but some of the
DNA looped out and was unpaired to the mRNA. (Fig. 16.5a)
e. Conclusion: The template DNA had stretches of nucleotides
that were not present in the mRNA, so they could not hybridize
and consequently looped out away from the mRNA. A 1:1 ratio
of nucleotides did not exist between the DNA and the mRNA.
(Fig. 16.5b)
5. W. Gilbert called the expressed portions of genes (translated
regions) exons and the intervening portions introns.
B. RNA splicing
1. Eukaryotic genes are first transcribed into a primary transcript that
contains both introns and exons. (Fig. 16.6a)
2. Intron splicing (removing the extra sequences) occurs inside the
nucleus.
3. Intron splicing is catalyzed by a complex of proteins and small
RNAs (snRNPsthat is, small nuclear ribonucleoprotein particles,
or snurps). (Fig. 16.6b, step 1)
a. snRNPs assemble on the primary mRNA transcript, forming a
spliceosome. (Fig. 16.6b, step 2),
b. The intron forms a loop that breaks when a specific adenine
nucleotide in the intron RNA attacks the 5' end of the intron.
c. The spliceosome mediates breakage of the intron at its 5' end,
and a loop forms. (Fig. 16.6b, step 3)
d. The loop is excised, and a phosphodiester bond links the exons
on either side. (Fig. 16.6b, step 4)
e. The excised intron is usually degraded to ribonucleotide
monophosphates.
C. Adding caps and tails to transcripts
1. A cap of 7-methylguanylate-P-P-P is attached to the 5' end of each
mRNA. (Fig. 16.7)
a. It serves as a recognition signal for the translational machinery.
b. It protects the transcript from degradation.
2. A tail, consisting of 100250 adenines (poly(A) tail), is added to
the 3' end of mRNA.
3. The coding sequence is flanked at the 5 and 3 ends by sequences
that are not destined for translation, untranslated sequences
(UTRs).
4. Caps and tails protect mRNAs from degradation by ribonucleases
and increase the efficiency of translation.

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Freeman, Biological Science, 4e, Chapter 16 Instructor Guide 5

5. Similarities and differences between eukaryotic and prokaryotic

transcription are summarized in Table 16.2.
III. An Introduction to Translation
A. Ribosomes are the site of protein synthesis.
1. Translation is the conversion of a sequence of nucleotides in an
mRNA into a sequence of amino acids in a protein.
2. Cells that synthesize lots of protein have lots of ribosomes, and
vice versa.
3. Studies showed that the basic mechanism of translation was the
same throughout the tree of life.
4. R. Britten and colleagues tested ribosomes as the site of protein
synthesis.
a. They conducted a pulse-chase experiment with 35S to label
proteins and track their locations at various times during and
after translation.
b. Results:
(1) Immediately after giving the pulse, the researchers found
radioactivity in ribosomes.
(2) Two minutes later, they found radioactivity only in free
proteins.
c. Conclusion: Protein synthesis occurs at the ribosome, and then
the protein is released from the ribosome.
B. Comparing translation in bacteria and eukaryotes
1. Electron microscopy of Escherichia coli DNA being transcribed
shows that:
a. Ribosomes have attached to mRNA even before transcription is
complete, while the mRNA is still linked to DNA.
b. Ribosomes translate mRNA as it is being synthesized by RNA
polymerase (transcription and translation occur simultaneously
in bacteria). (Fig. 16.8a, b)
c. Multiple ribosomes attach to each mRNA, forming a polysome.
d. Transcription and translation are physically connected because
there is no nuclear membrane.
2. In eukaryotes:
a. mRNAs are released from DNA in the nucleus.
b. mRNAs are then processed before leaving the nucleus.
c. Then they become associated with ribosomes in the cytoplasm
(transcription and translation are separate processes in
eukaryotes). (Fig. 16.9)
C. How does an mRNA triplet specify an amino acid?
1. Hereditary instructions are contained in sequences of nucleotides,
which are then converted into a sequence of amino acids.
2. Early hypothesis: Nucleotides of an mRNA codon chemically
combine with amino acid side chains through shape or charge
interactions. (Fig. 16.10a)

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Freeman, Biological Science, 4e, Chapter 16 Instructor Guide 6

3. Crick hypothesis: Adapter molecules hold amino acids in place

while interacting with an mRNA codon (predicted the existence of
transfer RNA molecules). (Fig. 16.10b)
IV. The Structure and Function of Transfer RNA
A. Requirements for translation
1. Ribosomes provide the machinery.
2. mRNAs contribute the message.
3. Amino acids are the building blocks.
4. ATP and GTP provide energy.
5. tRNA is important.
a. A tRNA that becomes covalently linked to an amino acid is
called an aminoacyl tRNA.
b. The addition of amino acids to tRNAs (charging a tRNA) is
catalyzed by aminoacyl tRNA synthetases. (Fig. 16.11)
c. Each of the 20 amino acids has a different aminoacyl tRNA
synthetase and one or more tRNAs.
d. Amino acids are transferred from aminoacyl tRNAs to proteins
synthesized in ribosomes. (Fig. 16.12)
B. What do tRNAs look like?
1. Sequencing revealed that tRNAs are 7585 nucleotides long.
2. Secondary structure was inferred from the nucleotide sequence.
(Fig. 16.13)
a. Hydrogen bonding occurs between complementary bases in
different parts of the tRNA molecule.
(1) Short, double-stranded regions form.
(2) The tRNA assumes a cloverleaf shape of stems and loops
(stems are double-stranded; loops are single-stranded
regions). (Fig. 16.14a)
b. All tRNAs have the sequence CCA at their 3' end; it is a binding
site for an amino acid.
c. One of the loops has a triplet of bases that varies in each tRNA;
it is the anticodon (i.e., the nucleotides that form base pairs with
an mRNA codon).
3. X-ray crystallography shows tertiary structurethe 3-D
arrangement of atoms.
a. The tRNA cloverleaf folds over, producing an L-shaped
structure.
b. Each tRNA has a distinct anticodon and amino acid.
c. The anticodon and amino acid attachment sites are separated
by a precise distance. (Fig. 16.14c)
d. Students should be able to make a rough sketch of a tRNA
with and without an amino acid attached, and explain the
relationship between the anticodon of a tRNA and a codon
in an mRNA.
C. How many tRNAs are there?
1. There are 61 different mRNA codons but only about 40 tRNAs.

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Freeman, Biological Science, 4e, Chapter 16 Instructor Guide 7

2. How can an mRNA codon, for which there is no tRNA, be

translated?
3. Cricks wobble hypothesis:
a. Many amino acids are specified by more than one codon.
b. Codons for the same amino acid tend to differ only in the third
position.
(1) Example: Codons 5'-CAA-3' and 5'-CAG-3' code for
glutamine.
(2) A tRNA with the anticodon 3'-GUU-5' can pair with either 5'CAA-3' or 5'-CAG-3'.
(3) The third codon position is the wobble position; it often
contains an inosine base that can pair with A, U, or C.
V. The Structure and Function of Ribosomes
A. Characteristics of protein synthesis
1. The sequence of bases in an RNA message is translated into a
sequence of amino acids in a polypeptide.
2. The conversion of each mRNA codon begins when the anticodon
of an aminoacyl tRNA binds to the codon.
3. The conversion is complete when a peptide bond forms between
the tRNAs amino acid and the growing polypeptide.
4. Conversion occurs inside a ribosome, which is composed of two
subunits. (Fig. 16.14)
a. There is a larger 50S subunit and a smaller 30S subunit.
b. Both subunits are composed of multiple RNA molecules and
numerous proteins.
c. The small subunit holds the mRNA in place during translation.
d. The large subunit is where peptide bond formation takes place.
e. The large ribosomal subunit has three tRNA binding sites. (Fig.
16.14a)
(1) During translation, the acceptor or aminoacyl (A) site holds
the tRNA with an amino acid that is about to be added to
the polypeptide.
(2) The peptidyl (P) site holds the tRNA that is holding the
growing polypeptide chain.
(3) The exit (E) site holds the empty tRNA that is about to be
released.
5. Three-step sequence of protein synthesis: (Fig. 16.15)
a. Aminoacyl tRNA diffuses into the A site; its anticodon binds to a
mRNA codon
b. A peptide bond forms between the amino acid on the aminoacyl
tRNA in the A site and the growing polypeptide (held by a tRNA)
in the P site.
c. The ribosome moves ahead, and all the tRNAs move one
position down the mRNA. The tRNA in the E site exits, the
tRNA in the P site moves to the E site, and the tRNA in the A
site moves to the P site.

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Freeman, Biological Science, 4e, Chapter 16 Instructor Guide 8

6. The polypeptide has grown by one amino acid.

7. The process occurs up to 20 times per second in bacteria and
about 2 times per second in eukaryotes.
8. Protein synthesis begins at the amino end (N-terminus) of the
protein and proceeds to the carboxy end (C-terminus).
B. Initiating translation
1. In bacteria, the 30S ribosomal subunit binds to a sequence on
mRNA about six nucleotides upstream from an AUG start codon.
2. The sequence to which the 30S subunit binds is the ShineDalgarno sequence and contains all or part of 5-AGGAGGU-3.
3. The initial interaction is mediated by initiation factors. In
eukaryotes, initiation factors bind to the 5 cap on mRNA.
4. An aminoacyl tRNA with N-formylmethionine (f-met) binds to the
AUG codon. (Fig. 16.15, step 2) In eukaryotes, the first amino acid
is normal methionine.
5. Initiation is complete when the large ribosomal subunit binds and
the tRNA-bearing f-met occupies the P site.
C. Elongation: extending the polypeptide (Fig. 16.14)
1. After the ribosome subunits join, a new aminoacyl tRNA binds to
the mRNA codon in the A site of the ribosome (aminoacyl site).
This reaction requires GTP.
2. A peptide bond forms between the carboxyl end of the Nformylmethionine amino acid in the P site and the amino end of the
amino acid in the A site.
3. Is the ribosome an enzyme or a ribozyme?
a. The active site of the ribosome is composed of rRNA, so the
ribosome is a ribozyme.
b. There is support for the RNA world hypothesis: Life began with
RNA molecules.
4. Moving down the mRNA (Fig. 16.14, step 3)
a. The tRNA in the P site moves to the E site (exit site).
b. The new peptidyl tRNA moves from the A site to the P site.
c. The empty tRNA is ejected from the E site.
d. Translocation requires energy in the form of GTP and
assistance from elongation factors.
e. Translocation of the mRNA occurs, moving it through the
ribosome in the 5 3 direction.
5. The three-part cycle of elongation (arrival of aminoacyl tRNA,
peptide bond formation, translocation) repeats over and over.
6. Once elongation is under way, additional ribosomes can bind to the
translational start site and begin translating.
D. Terminating translation (Fig. 16.17)
1. Three stop codons are included in the genetic code: UAA, UAG,
and UGA.
2. The ribosome reaches any one of these codons on an mRNA.
a. Then release factor proteins enter the A site.

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Freeman, Biological Science, 4e, Chapter 16 Instructor Guide 9

b. The completed polypeptide is released from the tRNA in the P

site.
c. The ribosome separates from the mRNA.
d. The two ribosomal subunits dissociate from each other, allowing
subunits to attach to the start codon of another message so that
translation can begin again.
E. Post-translational modifications
1. Folding
a. Protein shape is critical to function.
b. How does folding occur to produce the 3-D structure of an
active enzyme?
c. Thermodynamic hypothesis: Proteins fold into their most stable
energetic state.
d. Molecular chaperones facilitate protein folding.
2. Chemical modifications
a. Many proteins go through the ER and Golgi apparatus and have
sugar and/or lipid groups added.
b. Many proteins have phosphate groups added or removed
during their lifetimes. The addition or removal of this large
negatively charged group changes a proteins structure and
thus changes its function.
Chapter Vocabulary
To emphasize the functional meanings of these terms, the list is organized
by topic rather than by first occurrence in the chapter. It includes terms that
may have been introduced in earlier chapters but are important to the
current chapter as well. It also includes terms other than those highlighted in
bold type in the chapter text.
template strand
non-template strand
transcription
nucleoside
triphosphates
(NTPs)
initiation phase of
transcription
elongation phase of
transcription
termination phase of
transcription
transcription
termination
sequence
basal transcription
factors

5' cap (7methylguanylate-PP-P)

3' poly(A) tail
ribosomal RNA
messenger RNA
primary RNA
transcript
transfer RNA
exons
introns
snRNPs
RNA splicing
spliceosomes
RNA polymerase
sigma protein
holoenzyme

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core enzyme
RNA polymerase I
RNA polymerase II
RNA polymerase III
translation
transfer RNA (tRNA)
aminoacyl tRNA
aminoacyl tRNA
synthetase
peptidyl tRNA
anticodon
CCA sequence
X-ray
crystallography
ribosome
translation
elongation

Freeman, Biological Science, 4e, Chapter 16 Instructor Guide 10

translation
elongation factors
GTP
peptide bond
formation
ribozyme
translocation
polyribosomes
translation
termination
release factors
cell-free translation
system
promoters
TATAAT

10 box
TTGACA
35 box
upstream
downstream
TATA box
wobble hypothesis
inosine
large (50S) subunit
small (30S) subunit
peptidyl/P site
aminoacyl/A site
exit/E site

Shine-Dalgarno
sequence/ribosomal
binding site
AUG codon
N-formylmethionine
stop codon
translation initiation
translation initiation
factors
post-translational
modification
protein folding
molecular
chaperones
phosphorylation

Lecture Activities
What Makes a Firefly Glow?
Estimated duration of activity: 510 minutes
The website http://gslc.genetics.utah.edu/units/basics/firefly/ has a really fun
and interesting review of transcription and translation in the context of the
question What makes a firefly glow? If your classroom is computer
equipped, this would be a nice website to go through with your students as a
review.
An In-Class Demonstration: Translation
Estimated duration of activity: 15 minutes
This activity is especially suited to large lecture classes, particularly if there
is enough space at the front of the room to assemble sizable groups of
students. (You can scale down the activity presented here for smaller
classes or smaller rooms.)
This activity involves turning students into ribosomes, messenger
RNAs, protein factors, ribozymes, aminoacyl tRNAs, and aminoacyl
synthetases. You can assemble some relatively simple, inexpensive
identification labels in advance (suggestions follow), and you can get a large
number of students in the class directly involved in acting out the process of
translation. This activity may seem elementary to you, but students will
welcome the break from lecture and appreciate the chance to visualize a
complex biological process. If you dont have time to put the activity
together, assign it to a teaching assistant and have the TA direct the activity
during class.
Procedure:
1. Have 24 students come to the front of the room to form an mRNA
molecule. The 24 students should form a long, straight line facing the rest

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Freeman, Biological Science, 4e, Chapter 16 Instructor Guide 11

of the class. Start with the first student on the far left side (stage left) and
proceed to the right.
a. Give student 1 a card to hold up that says 5.
b. Leave student 2 unlabeled (this person is part of the mRNA 5' leader
sequence).
c. Give students 3, 4, 5, and 6 each a label to wear that is marked SD
(Shine-Dalgarno sequence).
d. Leave students 7 and 8 unlabeled (they are the rest of the 5' leader
sequence).
e. Label students 9, 10, and 11 as A, U, and G (respectively) to mark
them as the AUG codon, which specifies methionine.
f. Label students 12, 13, and 14 as C, C, and A (respectively) to mark
them as the CCA codon, which specifies proline.
g. Label students 15, 16, and 17 as G, C, and A (respectively) to mark
them as the GCA codon, which specifies alanine.
h. Label students 18, 19, and 20 as U, G, and A (respectively) to mark
them as the UGA stop codon.
i. Leave students 21, 22, and 23 unlabeled (they are the 3' trailer
sequence of the mRNA).
j. Give student 24 a card to hold up that says 3.
2. Next, have nine students come to the front of the room to form the 30S
subunit of the ribosome. These students should form a group in front of
the 5' end of the mRNA molecule (between the mRNA and the audience).
a. Three of the nine students will be the E site (label each with an E).
Have them stand at the extreme 5' end.
b. Three students will be the P site (label each with a P). Have them
stand just to the right of the E site.
c. Three students will be the A site (label each with an A). Have them
stand just to the right of the P site.
3. Have the 30S subunit students link arms and, as a group, bind to the
Shine-Dalgarno sequence in the 5' leader of the mRNA.
a. They can be assisted by a couple of students labeled IF (initiation
factor).
b. The 30S subunit students should pause at the Shine-Dalgarno
sequence and then spread out across the front of the mRNA so that
the three 30S students labeled P are positioned in front of the three
mRNA students labeled AUG.
4. Off to one side of the mRNA, some students may be labeled as various
transfer RNAs and others as aminoacyl synthetases. Each of them will
hold a card specifying a single amino acid.
a. The aminoacyl synthetase (labeled Met aa-synth) should give a card
that says MET to the person labeled tRNA-met.
b. The tRNA-met student, who is now holding the MET card, should go
behind the mRNA molecule, directly behind the students labeled
AUG.

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Freeman, Biological Science, 4e, Chapter 16 Instructor Guide 12

c. The tRNA-met student may be assisted in positioning by one or more

IFs.
5. Now, assemble a group of 11 students behind the mRNA molecule and
the tRNA-met student.
a. Student 1 closest to the 5' end of the mRNA should be unlabeled.
b. The next three students will be the E site.
c. The next three students will be the P site.
d. The next student will be labeled as the 23S ribozyme.
e. The next three students will be the A site.
f. One student at the 3' end will be unlabeled.
g. Have the students link arms in order and then line up so that the 50S
E site students are directly aligned with the 30S E site students, and
so forth. At this point, the ribosome is completely assembled on the
mRNA, and the tRNA-met student is positioned between the 30S and
50S P-site students.
6. Next, a person labeled tRNA-pro should pick up a card labeled PRO
from the appropriate aa-synth and go to the A site of the mRNA
molecule.
7. The individual on the 50S ribosome who is labeled 23S ribozyme then
forms a peptide bond between the card labeled MET and the card
labeled PRO (staple the cards together or have the ribozyme string them
together through previously punched holes in the cards).
8. After the peptide bond forms, the cards are handed to the person labeled
tRNA-pro.
9. Translocation: All of the mRNA students link arms and each takes three
steps to the left (i.e., in the direction of the 5' end).
a. The tRNA students should stay with the AUG and CCA sites on the
mRNA.
b. Students labeled EF (elongation factor) can assist the mRNA
movement.
c. When translocation is complete, the tRNA-met student should be in
the E site of the ribosome, and the tRNA-pro student (holding the
MET-PRO peptide) should be located in the P site.
10. The tRNA-met student now leaves the E site.
11. A student labeled tRNA-ala now picks up the ALA card from the
appropriate synthetase, enters the A site, and binds to the GCA codon,
where the ribozyme can form a peptide bond.
12. Repeat steps 710, until the UGA stop codon is positioned at the A site.
a. Students labeled RF (release factor) enter the A site, instead of a
tRNA.
b. Release the completed peptide (of three amino acids) from the tRNA
at the A site.
c. Last, the 50S ribosome subunit students all link arms and move as a
group away from the mRNA, as do the 30S students, to complete one
round of translation.

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Freeman, Biological Science, 4e, Chapter 16 Instructor Guide 13

Suggestions for labels:

Make the labels large (use 8.5-by-11-inch paper). Design them to be
attached to the persons chest using safety pins, tape, or some other
rapid, easy method.
For the cards that represent amino acids, use paper or stiffer cardboard,
keeping in mind that theyll need to be quickly and easily linked together.
Use different colors of papers to designate different items. All the labels
for students who will be part of the mRNA could be on red paper, the
amino acid cards could all be orange, the 30S ribosome labels could be
green, the 50S blue, and so on.
All labels and cards may be photocopied onto colored paper from a
master sheet, or printed from a computer with colored letters on white
paper or black letters on colored paper.
For the procedure as outlined in steps 112, youll need the following labels
and cards:
a. Labels designed to go on a persons chest
18 red labels for the mRNA students:
4 marked SD
3each one marked with either A, U, or G
3each one marked with either C, C, or A
3each one marked with either G, C, or A
3each one marked with either U, G, or A
2 unmarked red labels for the leader and trailer students
9 green labels for the 30S students:
3 marked E
3 marked P
3 marked A
11 blue labels for the 50S students:
2 unmarked
3 marked E
3 marked P
3 marked A
3 purple labels for the tRNA students:
1 marked tRNA-met
1 marked tRNA-pro
1 marked tRNA-ala
3 white labels for the aminoacyl synthetase students:
1 marked MET aa synth
1 marked PRO aa synth
1 marked ALA aa synth
Optional: Labels marked IF, EF, and RF (for initiation, elongation, and
release factors), if you choose to have students perform these roles.
b. Cards to represent amino acids
1 marked MET
1 marked PRO
1 marked ALA

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c. Cards for either end of the mRNA molecule

1 marked 5'
1 marked 3'
Discussion Ideas
Because the central focus of this chapter is the link between nucleic acids
and proteins, this might be a good time to consider the differences between
molecules that are catalysts (proteins and RNA) and templates (DNA and
RNA). Prions are unusual molecules because they seem to be able to
perform both functions. How are prions different? Where do they fit in? You
may want to pose these questions to the whole class, to small groups, as
challenge questions for pairs of students, or as thought problems to prepare
for a general discussion.
Question 1: What makes DNA and RNA good templates?
Possible answers:
DNA and RNA can act as blueprints for new synthesis from
monomers.
When DNA double strands open and beginning primers are
established, synthesis proceeds in a predictable way, making a
complementary copy of each existing strand.
The start complex that includes RNA polymerase binds to the
promoter region of a gene in the DNA sequence, and the RNA
molecule is synthesized to be complementary to the DNA strand that
follows the promoter position.
DNA and RNA have very predictable structures under ordinary
conditions.
Question 2: Why are proteins considered poor templates?
Possible answers:
Proteins cannot act as blueprints for new synthesis from monomers.
Proteins do not have complementary pieces that can act as a
template.
Proteins have complex, three-dimensional structures that can vary
drastically even under physiological conditions.
Proteins do not have a means to regularly transmit their blueprints
through generations.
Question 3: Why are proteins the best catalysts?
Possible answers:
Proteins have the ability to change three-dimensional shapes
drastically, often with very small differences in their primary structure.
Proteins have many variable R groups in their amino acids to bind to
other molecules in order for changes to occur.
Question 4: What do prions do?
Possible answers:
Prions are normal proteins in cells. Mutated or misfolded forms exist,
and they apparently can cause normal forms to assume the mutated
shape without intervention of a nucleic acid like RNA or DNA. (You

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Freeman, Biological Science, 4e, Chapter 16 Instructor Guide 15

could quiz students about how they could test thistreating

suspensions with DNase or RNase to see whether the mutated prions
still change normal ones.)
Cases have now been documented that somehow these mutated
prion forms present in food can affect normal prions in the brains of
those species exposed.
Prions seem to act as infectious agents but have no known nucleic
acid.
Question 5: Are these prions like catalysts or templates?
Possible answers:
Prions do not seem to be able to act as templates to form entirely new
molecules using only monomers.
Prions change shape like other proteins that act as catalysts. The
prions are misfolded compared to the normal protein.
Prions or some information related to them survive the digestive
system, absorption, and migration through the body to get to the site
where they do damage. The protein that has changed shape is
partially protease resistant.
The host does not mount an immune response, which suggests that
no virus is associated with the protein.
Some explanation needs to be found for how the first change in the
normal protein took place. The ability to change shape might be
possible, at least due to the flexibility of shape for proteins. After all,
they are good catalysts.
Although the differently folded proteins of the infectious particles are
called mutant, are they? Families with inherited forms of the human
disease all have mutations in their normal protein gene.
In England and France, these infectious agents have apparently
crossed the species barrier from cattle and sheep to humans. This
makes them even more mysterious. This transmission is different
from the inherited type; presumably, some sort of agent is
transmitted.
To emphasize the importance of in vitro experiments to biological
research, and to focus on the differences between transcription and
translation, you can present the following challenge question to the class.
You can also make a connection to DNA replication, which was discussed in
Chapter 14, by asking the question given in part c.
Question 6: How is a cell-free transcription system different from a cell-free
translation system?
a. What purified components would you need to combine to achieve
transcription in vitro?
Answer: Template DNA that contains one or more genes (with their
promoters)
RibonucleotidesATP, GTP, CTP, and UTP
RNA polymerase enzyme

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Freeman, Biological Science, 4e, Chapter 16 Instructor Guide 16

If the gene is prokaryotica cell fraction that contains sigma

factors
If the gene is eukaryotica nuclear extract that contains
transcription factors
b. What purified components would you need to combine to achieve
translation in vitro?
Answer: Ribosomes
Messenger RNA
Amino acids (all 20)
GTP and ATP to provide energy
A cell fraction that contains aminoacyl tRNAs, aminoacyl
synthetases, initiation factors, elongation factors, and
termination factors
c. How is an in vitro DNA replication system different from transcription and
translation systems? What components would be needed to achieve
replication in vitro?
Answer: Template DNA
DeoxyribonucleotidesdATP, dGTP, dCTP, and dTTP
DNA polymerase
A protein fraction from bacterial cells that contains helicases,
topoisomerases, single-stranded binding proteins, and
primase
Clicker Questions
1. Assume that RNA polymerase transcribes a gene containing the
following section of DNA:
5'-GATGCGAATCGT-3'
3'-CTACGCTTAGCA-5'
If the top strand were the antisense strand, the RNA corresponding to
this section would be _____.
1a.
2b.
3c.
4d.

5'-GATGCGAATCGT-3'
5'-GAUGCGAAUCGU-3'
5'-ACGATTCGCATC-3'
5'-ACGAUUCGCAUC-3'

Answer: 4d
Section Reference: 16.1
Blooms Taxonomy: Level 3 Application
2. Biologists discovered eukaryotic genes in pieces (split genes) in the late
1970s in experiments involving in vitro hybridization (base pairing)
between mRNA molecules and the DNA strands that act as templates for
their synthesis. What did biologists observe, and what did they conclude?

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Freeman, Biological Science, 4e, Chapter 16 Instructor Guide 17

1a. mRNADNA hybrids contain loops of single-stranded RNA; these

RNA loops represent introns.
2b. mRNADNA hybrids contain loops of single-stranded RNA; these
RNA loops represent exons.
3c. mRNADNA hybrids contain loops of single-stranded DNA; these
DNA loops represent introns.
4d. mRNADNA hybrids contain loops of single-stranded DNA; these
DNA loops represent exons.
Answer: 3c
Section Reference: 16.2
Blooms Taxonomy: Level 3 Application
3. A particular triplet of bases in the template strand of DNA is 5'-TGA-3'.
Which of the following is the anticodon component of the tRNA that binds
the mRNA codon transcribed from this DNA? (Note: By convention, the 3'
end of each anticodon is written on the left and the 5' on the right.)
1a. ACU
2b. AGU
3c. AGT
4d. UGA
Answer: 2b
Section Reference: 16.4
Blooms Taxonomy: Level 4 Analysis
4. Which of the following experiments would verify that ribosomes and
spliceosomes can be categorized as ribozymes?
1a. Add spliceosome complexes to a solution of DNA and compare to a
control solution with only DNA to see if mutation increases.
2b. Add spliceosome complexes to a solution of proteins and compare to
a control solution with only proteins to see if pH increases.
3c. Add spliceosome complexes to a solution of DNA and compare to a
solution of spliceosome complexes only to see if the sequence of
spliceosome subunits changes.
4d. Add complexes to a solution of pre-mRNA transcripts and compare to
a solution of pre-mRNA transcripts only to see if the reactions that
produce mRNA increase in rate.
Answer: 4d
Section Reference: 16.2 and 16.5
Blooms Taxonomy: Level 4 Analysis

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Freeman, Biological Science, 4e, Chapter 16 Instructor Guide 18

5. The template strand of DNA at the beginning of a protein-coding region

has the sequence: 5'-TACTGGGATAGCC*TACAT-3'. The * indicates the
position of a point mutation: A T originally present at this location has
been deleted. This deletion will most likely result in _____.
1a. mRNA codons preceding the mutation being misread
2b. mRNA codons following the mutation being misread
3c. no change in the polypeptide coded by this gene
4d. the AUG triplet functioning as a chain terminator
Answer: 2b
Section Reference: 16.6
Blooms Taxonomy: Level 3 Application

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