Chapter 14: Mechanisms and Regulation of Translation
I. Introduction
A. Introduction To Translation: What Is Translation?
1. In the process of gene expression, translation is the next major
step after
a. Transcription
b. Pre-mRNA processing
2. The goal of translation is to produce a protein
3. The protein is what is going to perform the function of the gene
a. Assuming proper folding
b. Assuming the proper modifications are made
B. Introduction To Translation: How The Mechanisms Of Translation
Were Discovered
1. Many aspects of the mechanism of translation were learned
through the study of how viruses worked
2. Viruses are composed of generally two types of molecules
a. Nucleic Acid
b. Protein
3. Viruses cannot reproduce on their own
a. Cannot replicate their own genetic material
b. Cannot express their own genes
4. Viruses use the host cell machinery to do both
a. Virus preferentially has the host cell translate its mRNAs
b. RNA biologists figured out how translation of cellular
mRNAs works
C. Introduction To Translation: The Importance of Translation
1. Translation is the most highly conserved process in nature
a. All viruses perform translation (not all viruses perform
transcription)
b. All organisms perform translation
2. The process of translation is an energetically costly process
a. In a rapidly growing bacterial cell, about 80% of a cell’s
energy is used for translation
b. 50% of a cell’s dry weight is dedicated to translation
3. The synthesis of a single protein requires the coordinated
action of over 100 proteins and RNAs
a. Some are part of the ribosome
b. Some are involved in binding the ribosome to mRNA
c. Some are involved in bringing amino acids to the site of
translation
II. RNAs That Are Required For Translation
A. RNAs That Are Required For Translation: Eukaryotic mRNA Structure
1. As defined by the presence of the start and stop codons, the
mRNA has three basic units
a. 5’ Untranslated Region (UTR)
b. Open Reading Frame (ORF)
c. 3’ Untranslated Region (UTR)
2. The 5’UTR:
a. Scanned by the 40S subunit of the ribosome
b. Has significant secondary structure-regulation
3. The open reading frame:
a. Also known as the protein coding region
b. Begins with the presence of a start codon on the 5’ end
and ends with a stop codon on the 3’ end
4. The 3’UTR:
a. Serves as a site of mRNA regulation
b. Has significant secondary structure
5. Supplemental Figure: RNAs That Are Required For Translation:
Eukaryotic mRNA Structure
B. RNAs That Are Required For Translation: Eukaryotic vs. Prokaryotic
mRNA
1. mRNAs can have multiple ORFs
a. Monocistronic messages (one ORF)
b. Polycistronic messages (multiple ORFS)
2. Eukaryotic mRNAs
a. Most are monocistronic
b. C. elegans does produce some polycistronic mRNAs
3. Prokaryotic mRNAs
a. Commonly are polycistronic
b. Ex. Lac operon
C. RNAs That Are Required For Translation: mRNA Reading Frames
1. In a eukaryotic mRNA the start codon is almost always the first
AUG
2. The start codon has three functions
a. Define the start site of translation
b. Specifies the first amino acid to be incorporated into the
new protein
c. Defines the reading frame for all subsequent codons
3. As we learned, the sequence of the mRNA is read 3 nucleotides
at a time (considered codons)
4. Each codon is immediately adjacent to, but not overlapping
with the next codon
a. Any stretch of mRNA could be translated in three
different frames (which would lead to greatly different
proteins)
b. The start codon will actually show us which of the three
frames is the correct one
D. RNAs That Are Required For Translation: Introduction To tRNA
1. tRNA molecules serve as the adaptors between the codons in
the mRNA and the amino acids they encode
2. “tRNA looks like nature’s attempt to make RNA do the job of a
protein” –Francis Crick (Cold Spring Harbor Symposium on
Quantitative Biology-1966)
3. RNA chains can fold into unique 3-Dimensional structures that
can act similarly to globular proteins
4. The 3-Dimensional (tertiary) structures found in RNA arise
from interactions between multiple secondary structural
domains
5. There have to be many different types of tRNAs, each encoded
by a separate gene
a. Creates multiple tRNA with different anti-codons
b. Each of these tRNA with different anti-codons bind
different codons
c. Each of these tRNA with different anti-codons are
charged with different amino acids
E. RNAs That Are Required For Translation: Basic tRNA Structure
1. The first structure published for the tRNA was the cloverleaf
secondary structure by R.W. Holley et. al.
a. Studied the tRNA for alanine
b. Contained three loops
c. Structure is incorrect
2. tRNA twists into an L-shaped 3-dimensional structure
a. Through X-ray crystallography
b. The “loops” from the cloverleaf structure are present as
stems in the actual L-shaped structure
F. RNAs That Are Required For Translation: tRNA Maturation and
Modified Bases
1. The pre-tRNA is produced and then undergoes processing to
get a mature tRNA
a. pre-tRNA is transcribed about twice the length of its
final form –
b. pre-tRNA transcript is processed by various nucleases at
both the 5’ and 3’ ends to produce a tRNA that is about 76
nt long
2. tRNA also must form complex tertiary structure to function
properly-to form tertiary structure:
a. Extensive base pairing
b. Incorporation of modified bases which help to increase
the amount of extensive base pairing within the tRNA
structure
3. tRNA can also contain more than 50 modified bases-types of
modifications
a. Methylation
b. Restructuring of the purine ring
4. Inosine (I) was the first modified base to be identified in tRNA
5. Pseudouridine (Ψ) is the most commonly seen modified base
in tRNA
a. The first modified nucleoside seen in any RNA
b. Pseudouridine has a restructured pyrimadine ring
G. RNAs That Are Required For Translation: tRNA Loops Have A
Separate Function
1. The defining of the “loops” came from the initial tertiary
structure-the cloverleaf
3. The tRNA has three loops
a. ΨU loop (T-loop)
b. D-loop
c. Anticodon Loop
4. The ΨU loop is involved in ribosome binding
5. The anticodon loop is involved in base pairing with the codon
in mRNA
a. Consists of the 3 nitrogenous bases
b. Is bounded by a uracil on the 5’ side and a modified
purine on the 3’ side
c. Each 3 base codon in the mRNA specifies a specific tRNA
which is charged with a specific amino acid
6. The D-loop:
a. Is recognition by the aminoacyl tRNA synthetases
b. “charge” the tRNA with the appropriate amino acid
H. RNAs That Are Required For Translation: Coaxial Stacking of Stems in
The L-Shaped tRNA Structure
1. The L-shaped structure is the true structure
a. Seen by X-ray crystallography
b. The loops we talked are really stems
2. The list of stems in the L-shaped tRNA
a. The 7 base pair acceptor stem stacks on the 5 bp T-stem
to form one continuous A-type helical arm of 12 bp
b. The D-stem and the anticodon stem also stack
3. The two sets of stacked stems give the tRNA its L-shaped
structure
4. Co-axial stacking is a common feature of RNA, for example in
rRNA co-axial stacking of as many as 70 base pairs can be found
J. RNAs That Are Required For Translation: L-Shaped Model of the tRNA
Structural Features
1. For each tRNA, the appropriate amino acid is directly bound to
the 3’ end of the tRNA
a. Every tRNA have the sequence 5’-P-CCA-OH-3’ at the far
3’ end of the tRNA
b. Correct amino-acyl tRNA synthetase will charge the
tRNA based on the anticodon sequence
2. Another commonly observed motif in the tRNA is the U turn
3. The U-turn is caused by hydrogen bonding of the N3 position of
uridine with the phosphate group of a nucleotide three positions
downstream
K. RNAs That Are Required For Translation: Attachment of Amino Acids
To The tRNA
1. Overall, there are two classes of amino-acyl tRNA synthetases
a. Class I enzymes which attach the amino acid to the 2’OH
of the final nucleotide of the tRNA and are monomeric
b. Class II enzymes which attach the amino acid to the 3’
OH of the final nucleotide and are dimeric or tetrameric
2. Each amino acid has its own amino-acyl tRNA synthetase
L. RNAs That Are Required For Translation: Attachment of Amino Acids
To The tRNA
1. When a tRNA becomes bound to an amino acid, an acyl linkage
is formed between the carboxyl group of an amino acid and
either the 2’ or 3’ OH group of the final nucleotide (which is an
adenine)
2. The acyl linkage is considered a high energy bond
a. Hydrolysis of this bond results in a large change in free
energy
b. Energy released when this bond is broken during
translation helps drive formation of a peptide bond
between amino acids
3. The process of creating the acyl linkage between the tRNA and
the amino acid occurs in two enzymatic steps
a. Adenylylation (transfer of AMP), in which the amino
acid reacts with ATP to become adenylated with the
release of pyrophosphate
b. tRNA charging in which the adenylylated amino acid
reacts with the tRNA resulting in a transfer of the amino
acid to the 3’ end of the tRNA via either the 2’ or 3’ OH
group of the final nucleotide and a release of AMP
III. The Ribosomome
A. The Ribosome: An Introduction
1. The ribosome is a macromolecular machine that directs the
synthesis of proteins
2. The ribosome consists two subunits in both prokaryotes and
eukaryotes
a. Small Subunit
b. Large Subunit
3. The ribosome has an overall molecular weight of greater than
2.5 megadaltons
a. At least 3 different rRNAs
b. More than 50 different proteins
4. Compared to DNA and RNA polymerase II, the ribosome
actually works quite slowly
a. Adds only 2-20 amino acids per second
b. DNA polymerase can add 200-1000 nucleotides per
second
c. RNA polymerase II can transcribe 30-60 nucleotides per
second
5. Subcellular localization of ribosomes
a. In eukaryotes in the cytoplasm (transcription and
translation are separated)
b. In prokaryotes-free floating in the cell ((cotranscriptional)
B. The Ribosome: Eukaryotic vs. Prokaryotic Ribosome Structure
1. Each ribosomal subunit has a particular function
a. The large subunit which contains the peptidyl
transferase center which is responsible for formation of
peptide bonds
b. The small subunit which contains the decoding center in
which charged tRNAs decode the information in the mRNA
codon by codon
2. By convention, the large and small subunits are named
according to their velocity of sedimentation when centrifuged
3. As measured by Svedbergs (S), the prokaryotic ribosome
sediments the following way
a. Small subunit sediments at 30S
b. Large subunit sediments at 50S
c. Total ribosome sediments at 70S
4. As measured by Svedbergs (S), the eukaryotic ribosome
sediments the following way
a. Small subunit sediments at 40S
b. Large subunit sediments at 60S
c. Total ribosome sediments at 80S
C. The Ribosome: RNA Composition of The Large and Small Subunits
1. Like the full subunits, the rRNAs are distinguished by the
centrifugal speed at which they sediment in a gradient
2. The small subunits contain the following rRNAs and proteins
a. Bacterial 30S subunit contains the 16S rRNA and 21
proteins
b. Eukaryotic 40S subunit contains the 18S rRNA and 33
proteins
3. The large subunits contain the following rRNAs and proteins
a. Bacterial 50S subunit contains the 5S rRNA and 23S
rRNA and 34 proteins
b. Eukaryotic 60S subunit contains the 5S rRNA, the 5.8S
rRNA and the 28S rRNA as well as 49 proteins
D. The Ribosome: Functions of rRNA
1. Locations of rRNA and proteins in each each ribosomal subunit
a. rRNA located at the core-suggests important role
b. Proteins at the periphery
2. Main functions of the rRNA in the large subunit
a. Scaffold from which to build the larger superstructure of
each subunit
b. May have catalytic function as the peptidyl transferase
center in the large subunit is composed almost entirely of
rRNA
3. rRNA function in the small subunit
a. Anticodon loops of the charged tRNAs contact the 16S
rRNA in bacteria
b. The small subunit interacts with the mRNA through the
16S rRNA
E. The Ribosome: tRNA Binding Sites In The Small Subunit
1. The small subunit (in both prokaryotes and eukaryotes) has a
total of three sites for binding tRNAs
a. A-site
b. P-site
c. E-site
2. The A-site functions to bind the amino-acylated tRNA
3. The P-site functions as the binding site for the peptidyl-tRNA
(tRNA which holds the growing peptide)
4. The E-site is the binding site for the uncharged tRNA that will
exit after transfer to the amino-acylated tRNA
IV. Eukaryotic Translational Initiation
A. Eukaryotic Translational Initiation: Introduction
1. The process of initiation allows for the start of translation
2. In order for translation to be initiated, three events must occur
a. The ribosome must be recruited to the mRNA
b. A charged tRNA must be placed in the P-site (This is the
only time a charged tRNA directly binds in the P-site
c. Positioning of the ribosome over the start codon (which
allows for determination of the reading frame)
3. The tRNA that is necessary for initiation in eukaryotes is
termed the Met-tRNAiMet
B. Eukaryotic Translational Initiation: The Ribosome Is Recruited To
The 5’ Cap
1. In eukaryotes, each subunit of the ribosome binds at separate
times
2. The first step eukaryotic initiation is creating of the 43S preinitiation complex
3. The two critical components of the 43S pre-initiation complex
are as follows
a. 40S ribosomal subunit
b. Met-tRNAiMet bound to eIF2-GTP which is considered the
ternary complex
4. The role of the eIF2-GTP is to place the Met-tRNAiMet in the Psite of the small subunit
5. Other eukaryotic initiation factors (eIFs) that are associated
with the 43S pre-initiation
a. eIF1
b. eIF3
c. eIF5
C. Eukaryotic Translational Initiation: The Ribosome Is Recruited To
The 5’ Cap
1. A series proteins are bound to the 5’MpppG cap that are
responsible for recruitment of the 43S pre-initiation complex
a. eIF4E
b. eIF4A
c. eIF4G
2. eIF4E is considered the cap binding protein and directly binds
the cap structure
3. eIF4G associates with the cap by binding eIF4E
4. eIF4A is a helicase that associates with both the mRNA as well
as eIF4G, and is thought to be able to unwind secondary structure
ahead of the ribosome during translation (not required)
5. eIF4G is involved in one other critical interaction when it
comes to translational initiation
6. eIF4G is able to bind the poly(A) binding protein (PAB)
a. PAB is associated with the 3’ end of the mRNA
b. This interaction is necessary for efficient translational
initiation
7. Several observations show this interaction is necessary
efficient initiation
a. Circularized mRNA have been visualized by atomic force
microscopy
b. Synthetic mRNAs containing a cap, but not a poly(A) tail
are not efficiently translated
c. Synthetic mRNAs containing a poly(A) tail, but not a cap
also are not efficiently translated
D. Eukaryotic Translational Initiation: The Small Subunit Scans The
mRNA To Find The Start Codon
1. The 43S pre-initiation complex will bind the mRNA at the 5’
MpppG cap
a. Through interactions with eIF4G
b. Creates the 48S pre-initiation complex
2. Once the 43S pre-initiation complex becomes associated with
the mRNA
a. Scan the mRNA in the 5’  3’ direction for the first start
codon
b. Scanning is an ATP dependent process that is stimulated
by the eIF4A RNA helicase
3. The start codon is recognized through base pairing between
the anticodon and the initiator tRNA
a. Base pairing between the 5’ AUG 3’ start codon and the
5’ CAU 3’ anticodon
b. Reason why initiator tRNA is part of the 43S preinitiation complex
E. Eukaryotic Translational Initiation: Start Codon Context Is Important
1. Recognition of the start codon by the small ribosomal subunit
is dependent on sequence context
2. The optimal sequence around the first 5’-AUG- 3’ for initiating
translation was discovered by Marilyn Kozak and is known as the
Kozak sequence
3. The Kozak sequence is as follows 5’-G/ANNAUGG-3’
a. The first base in the Kozak sequence needs to be a
purine and is 3 bases upstream from the first nucleotide in
the start codon
b. The final base in the Kozak sequence is a guanine
c. Not all mRNAs have this consensus sequence, but those
that do are more efficiently translated
4. The Kozak sequence is thought to guide the interaction
between the anticodon of the initiator tRNA and the start codon
of the mRNA
F. Eukaryotic Translational Initiation: Joining Of The Large Ribosomal
Subunit
1. As 43S pre-initiation complex reaches the start codon several
reactions must occur to allow 60S subunit joining (contains
peptidyl transferase center)
a. Correct base pairing between the initiator tRNA and the
codon changes the conformation of the 43S pre-initiation
complex
b. This change in conformation of the 43S complex results
in a change in conformation of eIF5 which then stimulates
eIF2 to hydrolyze its GTP to GDP
2. eIF2 hydrolysis of GTP results in the dissociation of the
following proteins
a. eIF2-GDP
b. eIF1
c. eIF3
d. eIF5
3. In the process of dissociation of several eIFs, eIF5B-GTP binds
the initiator tRNA
4. The role of eIF5B-GTP is to then stimulate the joining of the
60S ribosomal subunit
5. The joining of the 60S subunit results in the formation of the
80S ribosome
6. Once the 80S ribosome is formed, eIF5B-GTP hydrolysis is
stimulated leading to release of the rest of the translational
initiation factors
7. At this point the 80S initiation complex is formed
a. A full functional ribosome is now bound to the mRNA at
the start codon
b. The initiator tRNA is bound in the P-site
c. The start codon is positioned at the P-site
V. Translational Elongation
A. Translational Elongation: Introduction
1. Once the 80S ribosome is assembled, translation has been
initiated
2. The process of polypeptide synthesis is considered
translational elongation
3. During translational elongation two general events must occur
a. Peptide bonds must form between the carboxy terminal
amino acid residue in the growing peptide and the new
amino acid to be added
b. The ribosome must translocate
4. The ribosome incorporates 2-20 amino acids per second, and
so it moves about 2-20 codons per second along the mRNA
B. Translational Elongation: The Peptidyl Transferase Reaction
1. The reaction to form a new peptide bond is the peptidyl
transferase reaction and is catalyzed by the large subunit of the
ribosome
2. In the peptidyl transferase reaction requires two components
a. Amino-acylated tRNA
b. Peptidyl tRNA-3’ end is attached to the carboxyl
terminus of the growing peptide
3. To catalyze new peptide bond formation, the 3’ ends of the
amino-acyl and peptidyl tRNAs are brought in close proximity
a. Allows the amino group of the amino acid attached to
the amino-acyl tRNA to attack the carbonyl group of the
carboxy terminal amino acid attached to the peptidyl tRNA
b. Allows a new peptide bond to form
4. There are two important consequences of this method of
peptide synthesis
a. Allows for synthesis of the peptide in the amino to
carboxyl terminal direction
b. Allows transfer of the growing peptide from the peptidyl
tRNA to the amino-acyl tRNA
C. Translational Elongation: Introduction To Incorporation Of The
Correct Amino Acid
1. Translational elongation adds amino acids to the growing
peptide
2. Three events that occur to allow incorporation of the correct
amino acid
a. The correct amino-acyl tRNA must be placed within the
A-site of the ribosome (as directed by the codon which is
lying in the A-site
b. A peptide bond must be formed between the amino acid
linked to the tRNA in the A-site (amino-acyl tRNA) and the
growing peptide linked to the tRNA in the P-site (peptidyl
tRNA) through a peptidyl transferase reaction
c. The tRNA in the A-site must be translocated to the P-site
to allow for the next amino-acyl tRNA to bind
3. In addition, translocation will allow for a new codon to enter
the A-site
4. Unlike initiation, which requires many different proteins,
translational elongation only requires two proteins
a. eEF1 (composed of two subunits) (works like
prokaryotic EF-TU)
b. eEF2 (composed of multiple subunits) (works like
prokaryotic EF-G)
D. Translational Elongation: Delivering The tRNA To The A-site
1. eEF1 role in elongation:
a. Binds an amino-acylated tRNA
b. Brings amino-acylated tRNA to ribosome
2. eEF1-protein function is dependent on the state of the guanine
nucleotide bound to it
a. Guanine nucleotide state ensure that only a charged
tRNA enters the A-site
b. eEF1-GTP is capable of being bound to amino-acylated
tRNA
c. eEF1-GDP is not capable of being bound to aminoacylated tRNA
3. Any eEF1-GTP bound amino-acyl tRNA can enter the A-site of
the ribosome whether its anticodon base pairs with codon in the
mRNA or not
a. Attempted base pairing between anti-codon and codon
b. If an amino-acylated tRNA enters the A-site with the
wrong anticodon, it just dissociates from the A-site
4. A series of events happens once the amino-acylated tRNA with
the correct anti-codon base pairs with the codon,
a. First, base pairing between the anticodon and codon
occurs, eEF1 hydrolyzes its GTP to GDP
b. eEF1-GDP dissociates from the amino-acyl tRNA in the
A-site
c. eEF1-GTP hydrolysis is a way of “proofreading” or
ensuring the correct amino acid is added to the growing
peptide
d. Not the final proofreading mechanism
E. Translational Elongation: Another Proofreading Mechanism Exists
After eEF1 Is Released
1. To participate in the peptidyl transferase reaction, the tRNA
must rotate into the peptidyl transferase of the large subunit
(accomodation)
a. During the accomodation process, the 3’ end of the
amino-acylated tRNA moves about 70 Angstroms
b. Incorrectly base paired tRNAs cannot rotatate and
dissociate from the A-site (second proofreading
mechanism)
2. If the correct tRNA is in the A-site, it is able to appropriately
rotate
a. Allows the amino group of the amino acid to be added to
come close to the carboxy terminus of the growing peptide
bound to the tRNA in the P-site
b. The amino group attacks the carboxy group of the last
amino acid in the growing peptide chain allowing a new
peptide bond to form
c. At this point, the growing peptide is transferred to the
next tRNA
F. Translational Elongation: Translocation of The Ribosome
1. When a new peptide bond forms, the ribosome need to
translocate one codon 3’ on the mRNA
a. Moves the next codon into the A-site and causes the Asite of the ribosome to become empty
b. Moves the previous codon into the P-site (along with the
new peptidyl tRNA)
c. Moves the empty tRNA into the E-site
2. Translocation of both subunits does not happen at the same
time
a. The large ribosomal subunit which catalyzes peptide
bond formation translocates before the small ribosomal
subunit
b. This leads to the 3’ ends of each tRNA being shifted into
their new locations, but their anticodon ends are still in
their pre-peptide bond locations
3. The initial steps of translocation are coupled with the peptidyl
transferase reaction
4. eEF2 catalyzes translocation
a. Will only bind the ribosome in the GTP bound state
b. eEF2 has roughly has the same structure as a protein
bound tRNA
5. Steps in ribosome translocation
a. Large subunit shifts
b. The shift of the large ribosomal subunit will leave part
of the A-site in the large ribosomal subunit empty and
available for eEF2-GTP binding
c. Upon binding, GTP hydrolysis is stimulated
d. GTP hydrolysis causes a change in conformation of eEF2,
which allow it to enter the part of the A-site which is
present in the small ribosomal subunit, displacing the
tRNA present there
e. Upon entry of eEF2-GDP into the part of the A-site in the
small ribosomal subunit, translocation of the A-site tRNA
is triggered allowing the ribosome to fully move 1 codon 3’
along the mRNA
6. When translocation is complete, the ribosomal structure has
markedly reduced affinity for eEF2-GDP, which leads to its
release
7. Supplemental Figure: Translational Elongation: Translocation
of The Ribosome (Molecular Mimicry)
G. Translational Elongation: Forming A Peptide Bond Is An Energy
Dependent Process
1. Translational elongation is a very energy dependent process
2. For every peptide bond, the following is consumed
a. 1 molecule of ATP (used to charge the tRNA and create
the acyl linkage)
b. 2 molecules of GTP (during the elongation process)
3. Note: The energy from GTP is spent on ensuring accuracy
during the elongation process, and propelling the ribosome
down the mRNA in the 5’  3’ direction
VI. Translational Termination
A. Termination of Translation: Introduction
1. Termination of translation occurs when a stop codon enters
the A-site of the ribosome
a. UAA
b. UAG
c. UGA
2. The two events necessary for termination to occur
a. Release of the peptide
b. Dissociation of the ribosome from the mRNA
3. The proteins that are involved in peptide release are
considered release factors (RFs) and there are two classes of
these
a. Class 1 release factors decode the stop codons
b. Class 2 release factors are GTPases that stimulate the
activity of the class 1 release factors
B. Termination of Translation: The Mechanism
1. Termination begins when the stop codon enters the A-site
2. Note: There is no tRNA that recognizes a stop codon
3. Instead, the stop codon is recognized by eRF1
a. Is a class 1 factor that is bound to GTP
b. Can recognize all stop codons
4. Once eRF1 recognizes the stop codon, eRF3 mediates
hydrolysis of the eRF1-GTP to eRF1-GDP
a. Hydrolysis of the acyl linkage between the carboxy
terminal amino acid and the peptidyl tRNA
b. Release of eRF1 from the ribosomal A-site
c. Mechanism of action is unknown
VII. Regulation of Translation
A. Regulation of Translation: Introduction
1. Gene expression can still be controlled even if an mRNA is
produced, as it can be controlled at the level of translation
a. If an mRNA is translated then the gene is said to be
expressed
b. If an mRNA is not translated, then the gene is said to be
not expressed
2. In general, a significant amount of regulation occurs at the
transcriptional level
3. However, regulation of gene expression at the translational
level has several advantages
a. Allows rapid responses to external stimuli
b. Allows for the rapid start of gene expression after
periods of dormancy
4. Translational regulation mostly occurs at the level of
initiation, such that synthesis of incomplete proteins does not
occur
B. Regulation of Translation: Introduction To The Regulators
1. Two types of molecules have the capability of functioning to
regulate translation
a. Proteins
b. RNA
2. The two types of RNA that function to regulate translation are
as follows:
a. miRNA (which directly repress translation of an mRNA)
b. siRNA (which repress translation of an mRNA by causing
its degradation)
3. More often than not, a translational regulator will bind the
mRNA in one of two places
a. 5’ Untranslated Region (5’ UTR)
b. 3’ Untranslated Region (3’ UTR)
4. Binding of a regulator to the 5’ UTR results in a direct
inhibition of translational initiation
a. Blocking 43S Pre-initiation complex binding
b. Blocking 40S Ribosome scanning
5. Binding of a regulator to the 3’ UTR results in inhibition of
translation in ways yet to be fully understood
C. Regulation of Translation: Regulation of Ferretin mRNA
1. Regulating iron levels in the human body is of critical
importance,as many proteins need to coordinate an iron ion to
function properly
a. Hemoglobin
b. Myoglobin
c. Oxidative phosphorylation enzymes
2. Low cellular levels of iron lead to a condition called anemia,
which is more common in women than in men
a. Fatigue
b. Weakness
c. Shortness of breath
d. Light headedness
e. Palpitations
3. Severe symptoms include
a. Chest pain (up to heart attack)
b. Dizziness
c. Fainting
d. Rapid Heart Rate
4. Common risk factors are:
a. Poor diet
b. Intestinal disorders
c. Menstruation
d. Pregnancy
e. Chronic conditions
5. Treatment involves diet changes and iron supplements
6. In response to iron levels, the translation of the ferritin mRNA
is regulated
7. The ferritin mRNA (and gene) encodes the ferritin protein
a. Ferritin protein is an iron binding protein
b. Ferritin protein is a major regulator of cellular iron
levels
c. Ferritin stores and releases iron in a controlled manner
to maintain iron homeostasis in the cell
8. Translation of ferritin mRNA is regulated by a protein known
as the iron regulatory protein (IRP)
9. IRP binds an element within the 5’ UTR of the ferritin mRNA
called the Iron Response Element (IRE)
a. The IRE forms a hairpin loop
b. This hairpin loop must be resolved by eIF4A in order for
43S pre-initiation complex scanning to occur
10. The ability of the IRP to recognize the IRE is controlled by the
levels of iron in the cell
11. Under conditions of low concentrations of iron:
a. Iron concentration is too low and iron cannot bind the
IRP
b. IRP is able to bind the IRE and inhibit the ability of
eIF4A to unwind the IRE hairpin structure, which in turn
blocks the progression of the 43S pre-initiation complex
from scanning the ferritin mRNA for the start codon
12. Under conditions of high concentrations of iron
a. Iron concentration is high, and can bind the IRP
b. IRP, when bound to iron cannot bind the IRE
c. eIF4A can resolve the IRE secondary structure allowing
43S pre-initiation complex scanning
d. Ferritin mRNA translation
e. Ferritin protein acts to reduce iron levels by storing it
D. Regulation of Translation: Introduction To Global Regulators of
Translation
1. Besides being able to regulate the translation of a single mRNA,
translation of mRNA can be regulated in a global manner (almost
all mRNA present in the cell)
2. In general, translation is globally regulated in response to two
conditions
a. Reduced nutrients
b. Physiological stress
3. In these instances two early steps in translational initiation
are targeted for inhibition
a. Recognition of the mRNA by the 43S pre-initiation
complex
b. Initiator tRNA binding to the 40S ribosomal subunit
E. Regulation of Translation: Global Regulation of Translation-eIF2
Phosphorylation
1. One common mechanism to globally inhibit translation is to
phosphorylate eIF2
a. eIF2-GTP bind the initiator tRNA and delivers it to the Psite of the 40S ribosomal subunit
b. eIF2-GDP cannot bind the initiator tRNA
2. eIF2 is a multisubunit protein (heterotrimer)
a. α subunit
b. Β subunit
c. γ subunit
3. Phosphorylation of the α subunit is mediate by several kinase
enzymes
4. eIF2α kinases are activated in response to amino acid
starvation, viral infection and elevated temperature
5. Phosphorylation of the α subunit inhibits the action of a GTPexchange factor for eIF2 called eIF2B
a. Exchanges the GDP for GTP
b. Allows eIF2 protein to engage in another round of
translational initiation
F. Regulation of Translation: Global Regulation of Translation-eIF4E
1. To globally inhibit translational initiation the cell can target
the 5’ cap binding protein eIF4E
a. eIF4E is required to build a cap binding complex
including eIF4A and eIF4G
b. eIF4G is necessary for mRNA circularization and 43S
pre-initiation complex binding
2. The domain that eIF4G uses to bind eIF4E is also found in a
small family of proteins known as eIF4E binding proteins (4EBPs)
3. The 4E-BPs act to inhibit translation globally by competing
with eIF4G for binding to eIF4E
4. The activity of 4E-BPs, like many other proteins, is regulated
by a phosphorylation cycle
a. Unphosphorylated 4E-BPs have the ability to bind eIF4E
tightly, thus blocking in the ability of eIF4G to bind and
stimulate translation
b. Phosphorylated 4E-BPs do not have the ability to bind to
eIF4E
5. Phosphorylation of 4E-BPs is mediated by a key cellular
protein kinase called mTOR
6. The mTOR is activated in response to a receipt of a specific
signal
a. Growth factors
b. Hormones
c. Factors that stimulate cell division
7. In response to these signals mTOR phosphorylates 4E-BPs
leading to increased translational capacity in the cell
8. Given the fact that mTOR phosphorylation leads to increased
translational capacity and increased cell division, mTOR activity
can lead to cancer formation
9. Rapamycin is an mTOR inhibitor and an affective
chemotherapy agent
G. Regulation of Translation: 4E-BPs Can Regulate Translation of
Specific mRNAs
1. One 4E-BP named CUP regulates translation of the Oskar
mRNA during Drosphila development
2. The Oskar mRNA has elements in its 3’ UTR called Bruno
Response Elements (BREs) which are bound by the Bruno
protein
3. Bruno in turn acts to recruit CUP protein
4. The CUP protein interacts with eIF4E to block the binding of
eIF4G