Chapter
e13
Information in
Messenger RNA is
Used to Make Protein��������e250
The Genetic Code���������������e250
Proteins are Made
by Ribosomes����������������������e251
Translation in Bacteria
versus Eukaryotes��������������e252
Export and Location
of Proteins����������������������������e254
Many Antibiotics Target
Ribosomes����������������������������e254
Rare Amino Acids Encoded
by Stop Codons�������������������e254
Protein Degradation�����������e255
e250
Protein Synthesis
Information in Messenger RNA is Used to Make
Protein
Translation is the process by which information carried in the mes-
senger RNA (mRNA) is translated into a sequence of amino acids
called a polypeptide by ribosomes. Out of the three main types of
RNA, mRNA is the only type that is ever translated into protein. Both
ribosomal RNA (rRNA) and transfer RNA (tRNA) function as RNA
molecules in protein synthesis. The rRNA is part of the structure of
a ribosome and catalyzes the chemical reaction between two amino
acids. The tRNA carries amino acids to the ribosome. The details of
translation are discussed in this chapter’s Key Concepts below.
l Polypeptide chains are linear polymers of amino acids. There are
20 common genetically encoded amino acids plus two rare ones.
A polypeptide chain is produced from covalently linking the
monomers of proteins, amino acids. There are twenty amino acids that
are categorized based on their chemical and physical properties. The
specific property of the amino acid is dictated by the variable group
(R group) present, which may be acidic or basic, hydrophilic or hydro-
phobic, and charged or uncharged. There are other groups present in
amino acids besides R groups. These groups include an amino group,
a carboxyl group, and a hydrogen, which surround the central carbon.
The polypeptide chain folds into a 3 dimensional structure based upon
the chemical properties of its amino acids. Each amino acid is linked
to the next by the ribosome during translation. The covalent bond
between the amino acids is called a peptide bond.
The Genetic Code
l Each amino acid is encoded by a codon consisting of three
bases. Each codon is recognized by the corresponding anticodon
on a tRNA to which the amino acid is attached.
During translation, the bases on the mRNA are read in groups
of three called codons. Each codon is recognized by the anticodon
Molecular Biology, Second Edition Study Guide.
© 2013 Elsevier Inc.
Academic Cell is an imprint of Elsevier Inc.
 Proteins are Made by Ribosomes e251

portion of a tRNA that carries a specific amino acid. The anticodon on the tRNA is
complementary to the codon on the mRNA.
There are 64 possible codons, with each codon specific to an amino acid, but the
same amino acid may be encoded by multiple codons. Therefore, the genetic code is
redundant. Only 61 of the 64 possible codons encode amino acids. The other three
codons are stop codons and signal the ribosome to stop translating the mRNA.
For the most part, the genetic code is universal and shared by almost all organisms.
The exceptions being some protists, mycoplasmas, and animal/fungal mitochondria.

Bilbille Y et al. (2011) The human mitochondrial tRNAMet: structure/
function relationship of a unique modification in the decoding of unconven-
tional codons. J. Mol. Biol. 406:257–274
The mitochondria generates a majority of the ATP used in cellular
processes through oxidative phosphorylation. The genome of the human mitochondrion
contains 13 components involved in the electron transport chain and ATP synthase and
are synthesized exclusively within the organelle. Additionally, 22 tRNAs (mtRNAs) are
encoded within the mitochondrial genome. Each tRNA is designated for one amino
acid, with the exception of lysine and serine, each receiving two tRNAs. Codon reas-
signments are common in mitochondrial translational systems. The universal stop
codon, UGA codes for tryptophan. Additionally, AGA and AGG that normally code for
arginine, are used as stop codons in mammalian mitochondria. Of specific interest to
the authors of this paper is the AUA codon, which encodes isoleucine in the universal
code. Surprisingly, this codon is used to specify methionine for translation initiation and
elongation in mitochondria. In addition, the tRNA that recognizes the AUA codon con-
tains an anticodon that reads CAU, which means a non-standard C-A bond forms when
the tRNA and mRNA complex. The C-A bond should be unfavorable, yet the AUA is still
translated as a methionine. The authors set out to investigate why this occurs.
The authors suggest that a post-transcriptional modification at the
wobble position (third base position) might play a role in the recognition of the AUA
and AUG codons. The researchers in this associated paper focused on the folding, struc-
ture, and codon-binding properties of the human mitochondrial tRNA containing the
anticodon CAU, but still coding for methionine. They designated this as hmtRNAMetCAU.
Nuclear magnetic resonance (NMR) was utilized to examine the struc-
ture of hmtRNAMetCAU and the contribution of the post-transcriptionally modified cytidine at
position 34 on the tRNA anticodon loop. They were able to determine that the anticodon
wobble position modification, that is the modified cytidine, enhanced the AUA ribosomal
A-site codon recognition. The post-transcriptional modification of the wobble base of the
anticodon also enabled the tRNA to recognize other codons besides AUG and AUA. In addi-
tion to those two, the tRNA recognized AUU and AUC with about the same affinity as AUA.
In conclusion, the hmtRNAMetCAU containing the modified cytidine
residue at the wobble base is able to bind both AUA and AUG codons for initiation
and during elongation to translate those codons into methionine.
FOCUS ON
RELEVANT RESEARCH
Conceptual questions
1. How is the mRNA read to yield the individual amino acids?
2. What is the wobble base and how does it affect protein sequence?
3. Why is the genetic code unambiguous but redundant?
Discussion points
1. The authors discuss the role of the wobble base in gene expression. Post-
transcriptional modification of the anticodon on some tRNAs can yield changes
in the gene expression pattern, such as changes to coding of the start and stop
codons. How can these changes affect protein structure/function? What is the
evolutionary significance of this?

Using tRNAs that recognize the codons on mRNA, the appropriate amino acid
can be placed in the correct sequence in the polypeptide. In some cases, tRNAs can
read multiple codons but still carry the same amino acid. Transfer RNAs carrying
amino acids are called “charged” tRNAs. After the tRNA has given up its amino acid
at the ribosome, it leaves the ribosome as an uncharged tRNA. Aminoacyl tRNA
synthetases are enzymes that recharge the tRNAs.

Proteins are Made by Ribosomes

l Translation is the synthesis of proteins using information from RNA and is car-
ried out by the ribosome.

As discussed above, translation synthesizes proteins using the information in RNA.

The ribosome is the cellular machine that coordinates the translation of mRNA using
tRNAs as the “middle man” between nucleic acid sequence and amino acid sequence.
Ribosomes are made of rRNA and several proteins. The bacterial ribosome
(70S) contains two subunits: the large subunit (50S) and the small subunit (30S).
e252 CHAPTER THIRTEEN • Protein Synthesis

The large subunit contains the 23S and 5S rRNA while the small subunit contains the
16S rRNA. The eukaryotic ribosome (80S) is slightly larger and contains a large subu-
nit (60S) and a small subunit (40S).
The enzymatic activity of the ribosome is called peptidyl transferase and in bacte-
ria is conferred on the 50S subunit by the 23S rRNA, which is why the ribosome is a
type of ribozyme.

l Codons can be read in three possible reading frames, thus it is important to

locate the correct start codon by using recognition sequences.

For any mRNA, three possible reading frames exist. In each case, a different
amino acid sequence can be derived. Fortunately, mRNA has recognition sequences
and punctuation built into the sequence.
In bacteria the small ribosomal subunit first recognizes a ribosomal binding
site, also called the Shine-Dalgarno sequence, within the 5' untranslated region (5'-
UTR) of the mRNA. Then, the small subunit translocates, or travels downstream,
until it finds the first AUG. Translation always begins at the start codon, AUG, and
then is read codon by codon until the ribosome reaches one of the three stop codons.
In bacteria, the start codon is recognized by an initiator tRNA which carries a spe-
cial methionine, called N-formyl-methionine (fMet). This special methionine always
begins the polypeptide chain in bacteria. Eukaryotes simply use unmodified methio-
nine to begin their proteins. After the initiator tRNA and small subunit bind to the
start codon, the large subunit joins in and begins translating the rest of the mRNA.

l During elongation of the polypeptide chain, the tRNA moves between three
separate sites on the ribosome.
l Translation is terminated when the stop codon is read by a protein known as

release factor. The ribosome subunits are then recycled.

The ribosome has three binding sites for tRNAs: E (exit), P (peptide), and
A (acceptor). Once tRNAs have given up their amino acids and are uncharged, they
leave the ribosome through the E site. The A site accepts incoming charged tRNAs
and the growing polypeptide chain resides in the P site. The initiator tRNA begins
in the P site. The next codon downstream resides in the A site, where the incoming
charged tRNA binds. The peptidyl transferase activity of the ribosome catalyzes pep-
tide bond formation between the adjacent amino acids. Once fMet is bound to the
second amino acid, it no longer binds to its tRNA. The ribosome translocates (facili-
tated by elongation factors) towards the 3' end of the mRNA by one codon. This
places the growing polypeptide chain in the P site, the uncharged initiator tRNA in
the E site, and leaves the A site empty. The uncharged initiator tRNA exits the ribos-
ome and the tRNA carrying the third amino acid in the sequence binds to the A site,
and the process repeats itself until a stop codon is reached in the A site.
Placement of a stop codon in the A site induces release factors to bind to the
ribosome. The polypeptide chain is released from the ribosome and the ribosomal
subunits dissociate from the mRNA. All of the components are free to be used again.

l Several ribosomes usually read the same mRNA simultaneously.

Polyribosomes (Polysomes) form when multiple ribosomes are loaded onto the
same mRNA to translate it into protein. The simultaneous action of several ribosomes
quickly generates the protein, thus making the process both faster and more efficient.

Translation in Bacteria versus Eukaryotes

l In bacteria a single mRNA may encode several proteins. In addition, since
there is no nucleus, transcription and translation are coupled.
 Translation in Bacteria versus Eukaryotes e253

An operon is a cluster of genes that is transcribed as one long mRNA. Operons

are usually only present in bacteria. The coding sequence corresponding to each pro-
tein has its own Shine-Dalgarno sequence that ribosomes can bind. Consequently,
protein is made from each open reading frame (ORF) regardless of how many ORFs
are present on the mRNA.
Prokaryotes do not have a nucleus. Therefore, even before transcription of a pro-
tein coding gene has finished, ribosomes are already loading onto the transcript, and
translating the message into protein. This is called coupled transcription and translation.
Eukaryotes are not able to couple transcription and translation because of the nucleus.
Transcription occurs in the nucleus, and often, mRNA must be processed before it can
be translated. Translation occurs outside of the nucleus either on ribosomes in the cyto-
plasm or ribosomes embedded within the rough endoplasmic reticulum.

l There are differences between protein synthesis in eukaryotic and prokaryotic

cells.

Although the point of protein synthesis is to generate an amino acid sequence

from the information stored on mRNA, differences in mechanism and details exist
between prokaryotes and eukaryotes.
Prokaryotes utilize polycistronic mRNAs, that is, mRNA produced from operons.
Each open reading frame contains its own Shine-Dalgarno, so it is translated inde-
pendent of the other open reading frames on the same transcript. Operons are rare in
eukaryotes and mRNA directs the synthesis of only one protein, with rare exception.
Prokaryotes utilize a special methionine, called N-formyl-methionine (fMet),
for the first amino acid in a polypeptide chain. Eukaryotes just use the unmodified
methionine to begin protein synthesis.
Also, transcription and translation are coupled in prokaryotes because there is
no physical barrier separating the two processes in the cells. However, because of the
nucleus, transcription occurs in the nucleus and translation occurs in the cytoplasm.
Thus, transcription and translation are not coupled in eukaryotes.

Estaban M. (2009) Hepatitis C and evasion of the interferon system: a on mRNA and are therefore not subjected to initiation factors. Therefore, the virus
PKR paradigm. Cell Host Microbe 6:495–497. has blocked the defense system of the cell and at the same time, forced the cell into
translation of viral mRNAs.
The hepatitis C virus (HCV) affects 170 million people worldwide
and represents a significant health concern. This virus is responsible for chronic hepa-
titis and liver cirrhosis, along with hepatocellular carcinoma, a form of liver cancer.
No vaccine is available and control of the virus is through antiviral treatments with FOCUS ON
ribavirin and type I interferon (IFN). Often times, the virus stops responding to IFN RELEVANT RESEARCH
treatment. The mechanism for HCV evasion of IFN is not well understood. This paper
is a review summarizing the current research on IFN-resistance mechanisms of HCV.
Human hepatic cell lines Huh-7 infected with HCV were used to
study the IFN-evasion of the virus. The researchers gauged the effects of IFN on the Conceptual questions
infected cells by assaying for the presence of interferon-stimulated genes (ISGs).
1. How are eukaryotic 5’ caps needed for translation?
Cells with persistent infections produced fewer ISGs, which was deemed a post-tran-
2. What sequence do prokaryotes use to initiate translation?
scriptional effect due to the presence of ISG mRNA, suggesting that the virus blocks
3. How did HCV initiate transcription of its mRNAs?
translation. PKR is an inhibitor of translation and it was induced in IFN-treated HCV-
infected cells. Additionally, HCV induces the phosphorylation of PKR, which is respon-
Discussion points
sible for the reduced levels of ISG proteins in these cells. Since ISGs are important
for combating the virus, upon downregulation of ISGs, the virus is resistant to the 1. No vaccine for HCV infections exists. Additionally, chronic HCV infections are quite
antiviral effects of IFN. common, particularly for those individuals who have stopped responding to treat-
According to Figure 1 in the associated paper, HCV-IRES are able to ment. The treatment involves interferon and a viral replication inhibitor. How did
escape from PKR action by bypassing the need for functional translation initiation fac- the virus resist the antiviral effects of IFN? Why was it able to circumvent the
tors. The viral IRES are needed for translation initiation rather than eukaryotic 5’ caps effects of treatment?
e254 CHAPTER THIRTEEN • Protein Synthesis

The process of translation also contains many differences between the prokary-
otes and eukaryotes. Ribosomal binding sites, or Shine-Dalgarno sequences, are
present only in prokaryotes for initiation of translation. In eukaryotes translation ini-
tiation depends upon the 5' cap of mRNA. Additionally, the initiation, elongation, and
release factors are different between the two groups.
During the stationary phase of prokaryotes, or when resources are scarce, protein
synthesis slows due to inactivation of the ribosomes. Higher organisms inactivate ini-
tiation factors instead of ribosomes during periods of nutrient scarcity.

Export and Location of Proteins

l Proteins to be exported from the cell have signal sequences that direct them to
the export machinery.

Signal sequences are located on the N-terminus of some proteins and enable
those proteins to find their correct location outside the cell membrane. The signal
sequence tags the protein for transport through the cell membrane and out of the cell.
After transport, the signal sequence is cleaved off using a protease, an enzyme that
cuts proteins, and is not present in the mature protein.

l Molecular chaperones are specialized proteins that oversee the correct folding
of other proteins.

Chaperone proteins, or chaperonins, help direct the correct 3-dimensional shape of

other proteins. Chaperonins are classified as “holders” or “folders,” depending upon their
function. “Holders” prevent premature folding and “folders” help rectify misfoldings.

l Some proteins are made inside mitochondria and chloroplasts, but most
organelle proteins are made on cytoplasmic ribosomes and transported into
the organelles.

The Symbiotic Hypothesis argues that eukaryotic organelles, such as mitochon-

dria and chloroplasts, are likely derived from prokaryotes that were taken into early
eukaryotic cells and took up residence inside the cells instead of being used as a food
source. The prokaryotes conferred an advantage to the eukaryotic cells, either utili-
zation of oxygen in metabolism to yield more usable energy from nutrients (mito-
chondria) or the ability to make their own food (chloroplasts). Both mitochondria
and chloroplasts contain their own DNA, divide by binary fission, have their own
ribosomes, and produce their own proteins. All of these traits support the hypothe-
sis. Although organelles are able to make a few of their own proteins, many proteins
must still be transported into the organelles from the cytoplasm.

Many Antibiotics Target Ribosomes

l Many antibiotics inhibit protein synthesis. These include tetracycline,
aminoglycosides (such as streptomycin), and chloramphenicol.

Aminoglycosides bind to the 30S subunit of the bacterial ribosome. Streptomycin,

an aminoglycoside, binds to the 16S rRNA and inhibits binding of a tRNA into the A
site. Tetracyclines inhibit both bacterial and eukaryotic ribosomes by binding to the 16S
(prokaryotes) or 18S (eukaryotes) rRNA subunits and blocking attachment of charged
tRNAs. Ill effects are usually not observed in eukaryotes given tetracycline because
eukaryotic cells actively export tetracycline whereas prokaryotic cells import the anti-
biotic. Chloramphenicol binds to the 50S subunit and inhibits peptidyl transferase.

Rare Amino Acids Encoded by Stop Codons

l The rare amino acids selenocysteine and pyrrolysine are inserted at special
stop codons.
 Protein Degradation e255

The genetic code contains 20 amino acids, but many other amino acids are made
by modifying these 20 amino acids after proteins have been made. Selenocysteine and
pyrrolysine are rare genetically encoded amino acids. Selenocysteine is encoded by
one of the stop codons, UGA. Instead of the stop codon reading as such, it sometimes
encodes the rare amino acid. The choice between a stop codon and a selenocysteine
codon depends upon the presence of a selenocysteine insertion sequence (SECIS ele-
ment). In humans, 25 different genes encode proteins that use selenocysteine. Some
of these gene products are involved in combating oxidative stress. Pyrrolysine is also
encoded by a stop codon UAG, but only in a few genes of some Archaea.

Morley SJ and Willett M. (2009) Kinky binding and SECsy insertions. Mol. to selenium concentrations, prevents SBP2 from interacting with the SECIS elements
Cell 35: 396–398. and recoding the UGA codon. Therefore, UGA acts as a premature stop codon for
GPx1 and the protein is truncated and non-functional.
Selenium is supplied to the body through the diet. Several proteins
within the human body utilize selenocysteine, one of the rare amino acids. These are
called Sec-containing proteins and are often involved in combating oxidative stress
through antioxidants. When selenium is scarce in the body, the liver, kidneys, and FOCUS ON
lungs must decide which proteins that contain selenocysteine may continue to be syn- RELEVANT RESEARCH
thesized. This review summarizes the research into the investigation of how the cell
prioritizes the translation of Sec-containing proteins.
The authors discovered that a eukaryotic translation initiation factor,
4a3 (eIF4a3) suppresses translation by interacting with the 3’ untranslated region Conceptual questions
of target mRNAs. The introduction of Sec into the protein is done cotranslationally by
1. Which codons encode selenocysteine and pyrrolysine?
changing the stop codon, UGA, to a Sec-encoding codon, with the help of two tRNAs
2. What factor must be present in the mRNA for UGA to be recoded for seleno-
that recognize the UGA as a Sec codon, along with several other protein factors.
cysteine instead of functioning as a stop codon?
The expression of two Sec-containing proteins was examined. These
3. What types of proteins usually contain selenocysteine?
proteins included the essential housekeeping enzyme, phospholipid hydroperoxide
glutathione peroxidase (PHGPx), and the stress response enzyme, glutathione per-
Discussion points
oxidase 1 (GPx1). Both of these enzymes were present when selenium was abun-
dant. However, after limiting the selenium, the housekeeping PHGPx was synthesized 1. The SECIS element is present in the 3’ untranslated region of the mRNA for a
and any GPx1 mRNA present was quickly degraded. The production of Sec-containing Sec-containing protein. Several key proteins bind to this element to recode the
proteins is dependent upon the availability of a SECIS element in the 3’ UTR of tar- UGA stop codon into a selenocysteine codon. What do you think would happen if
get mRNAs. Several proteins, including Sec-specific elongation factors, recognize the the SECIS element was moved to another place within the same mRNA?
SECIS element, bind to it, and facilitate the binding of Sec-specific tRNAs to the UGA 2. The research group is also interested in the upregulation of eIF4a3 in response to
codon. Translational control is further moderated by binding of SBP2 to the SECIS selenium. Regulation of gene expression is not discussed until Chapters 16 and
element. The preference for translating PHGPx mRNA over GPx1 mRNA during sele- 17 of the textbook. However, using your current knowledge, what mechanism
nium scarcity occurs because high levels of eIF4a3, whose expression is sensitive do you think could be a plausible explanation for this phenomenon?

Protein Degradation
l Proteins are broken down by proteases. In eukaryotes proteins tagged with
ubiquitin are degraded in cylindrical assemblies called proteasomes.

Proteases, or proteinases, are enzymes that degrade proteins. The action of pro-
teases must be carefully controlled by the cell to prevent damage to non-targeted
proteins. Cells will often compartmentalize proteases to limit their involvement with
other cellular structures. Additionally, at least in eukaryotes, proteins are tagged with
ubiquitin, which is a signal for degradation. Once tagged, the proteins are fed into the
proteasome, which degrades the protein and cleaves off the ubiquitin tag. The tag is
recycled and attached to more targeted proteins.