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L I F E
S
C I
1a
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5’-UUA AUA UUC GAA
AGC UGC AUC GAA AAC UGU GAA UCA-3’
5’-TTA ATA TTC GAA
AGC TGC ATC GAA AAC TGT GAA TCA-3’
3’-AAT TAT AAG CTT
TCG ACG TAG CTT TTG ACA CTT AGT-5’
NOVEMBER 7, 2006
ROBERT A. LUE
Translation: the RNA-directed synthesis of proteins
1. The role of RNA in protein synthesis
a) Three classes of RNA are required to synthesize proteins
b) mRNAs are decoded in sets of three nucleotides
c) The structure and function of transfer RNA
d) Proofreading by aminoacyl-tRNA synthetase
2. The translation machinery and cycle
Lecture Readings
Alberts: pp. 243-255
McMurry: pp. 816-823
Transfer RNAs match amino acids to codons
2 key domains
Anticodon: Nucleotide
triplet that base pairs
with the complementary
codon in mRNA
3’-end: Attachment site
for the appropriate amino
acid
Transfer RNA (tRNA) are molecular adaptors that connect specific
amino acids with their matching codons
Some tRNAs recognize more than one codon by tolerating mismatch
base pairing at the 3rd position of the codon
Some amino acids are matched by more than one tRNA
The translation of mRNA into protein depends on adaptor molecules that can recognize and bind both to the codon and, at another site on their
surface, to the appropriate amino acid. These adaptors consist of a set of small RNA molecules known as transfer RNAs (tRNAs), are each about 80
nucleotides in length, and are synthesized by RNA polymerase III in eukaryotes.
Earlier this term we saw that RNA molecules can fold up into precisely defined three-dimensional structures, and tRNA molecules provide a
striking example. Four short segments of the folded tRNA are double-helical, producing a molecule that looks like a cloverleaf when drawn
schematically. The tRNA is further folded to form a compact L-shaped structure that is held together by additional hydrogen bonds between
different regions of the molecule.
Two regions of unpaired nucleotide sequence situated at either end of the L-shaped molecule are crucial to the function of tRNA in protein
translation. One of these regions forms the anticodon, a triplet nucleotide sequence that pairs with the complementary codon in an mRNA
molecule. The other is a short single-stranded region at the 3’ end of the molecule which is where the amino acid that matches the codon is
covalently attached to the tRNA.
You know that there is redundancy in the genetic code since several different codons can specify a single amino acid. This implies either that there
is more than one tRNA for many of the amino acids or that some tRNA molecules can base-pair with more than one codon; both situations occur.
Some amino acids are matched with more than one tRNA. The exact number of different kinds of tRNA differs from one species to the next.
Humans have 497 tRNA genes but, among them, only 48 different anticodons are represented. On the other hand, some tRNAs require accurate
base-pairing only at the first two positions of the codon and can tolerate a mismatch (or wobble) at the third position. This wobble base-pairing
explains why so many of the alternative codons for an amino acid such as threonine differ only in their third nucleotide.
Aminoacyl-tRNA synthetase couples each
tRNA with the appropriate amino acid
20 different aminoacyl tRNA synthetases in eukaryotes
Each recognizes one amino acid and all of its matching tRNAs
Before the codon or codons specifying a given amino acid can be recognized by a particular tRNA, the appropriate amino acid must be covalently
coupled to that tRNA. This essential process is catalyzed by an enzyme called aminoacyl-tRNA synthetase. Each of the 20 different synthetases
recognizes one amino acid and all its compatible tRNAs. These coupling enzymes link an amino acid to the free 2’ or 3’ hydroxyl of the adenosine
at the 3’ terminus of tRNA molecules. ATP hydrolysis is required for this reaction to occur, and the amino acid is linked to the tRNA by a highenergy bond. tRNAs that are conjugated with an amino acid are often described as being “charged” and the combination is referred to as an
aminoacyl-tRNA. As we will see later, the energy of the bond between the amino acid and the tRNA subsequently drives the formation of peptide
bonds between adjacent amino acids in a growing protein chain.
Aminoacyl-tRNA synthetase ensures that the correct
amino acid is coupled with the correct tRNA
Two sites (pockets) on tRNA synthetase proofreads the amino acid
•First, the synthesis site excludes amino acids that are too large
•Second, the editing site excludes the correct amino acid, but accepts
and removes incorrect amino acids that are similar in size
Two step editing results in a low error rate of 1 in 40,000 tRNA couplings
To ensure proper protein function the correct sequence of amino acids must be linked together during the course of translation. The first step
towards ensuring translational accuracy depends on the tRNA synthetase which is responsible for linking the correct amino acid to each tRNA.
Most synthetases select the correct amino acid by a two-step mechanism that involves two discrete sites on the enzyme. First, the correct amino
acid has the highest binding affinity for the active-site pocket (synthesis site) of its synthetase and is therefore favored over the other 19. Amino
acids that are larger than the correct one are excluded from the active site based on size. However, this mechanism for proof-reading the candidate
amino acid is not sufficient since distinguishing two amino acids of similar size is not possible at this site. For example, isoleucine and valine
differ by only a single methyl group and both are likely to fit into the synthesis site of the synthetase meant to only load isoleucine on the
appropriate tRNA.
Fortunately, a second proof-reading step occurs after the candidate amino acid has associated with the synthetase and the tRNA. In this step the
candidate amino acid is shifted into a second pocket in the synthetase. The precise dimensions and shape of this second site excludes the correct
amino acid but allows access by closely related amino acids. Once an amino acid enters this editing site, it is hydrolyzed from the tRNA and
released from the enzyme. This hydrolytic editing is analogous to the editing by DNA polymerases and increases the overall accuracy of tRNA
charging to approximately one mistake in 40,000 couplings.
The tRNA synthetase also recognizes the correct set of tRNAs based on their structural and chemical characteristics. Most tRNA synthetases
directly recognize the matching tRNA anticodon while others recognize the nucleotide sequence of the 3’ stem. Thus, several specialized sites on
the synthetase will recognize and bind nucleotides at several positions on the tRNA.
Translation: the RNA-directed synthesis of proteins
1. The role of RNA in protein synthesis
a) Three classes of RNA are required to synthesize proteins
b) mRNAs are decoded in sets of three nucleotides
c) The structure and function of transfer RNA
d) Proofreading by aminoacyl-tRNA synthetase
2. The translation machinery and cycle
a) The structure of the ribosome
b) The protein translation cycle
c) Starting and stopping translation
Lecture Readings
Alberts: pp. 243-255
McMurry: pp. 816-823
The structure of the Ribosome
Each ribosome is composed of two subunits: Large and Small
The ribosome is 2/3 RNA (4 RNAs) and 1/3 protein (>80 proteins)
RNAs are responsible for promoting the chemical reactions of translation
3 sites bind to tRNA:
A-site binds aminoacyl tRNA; P-site binds peptidyl tRNA; E-site binds exiting tRNA
We have just seen how tRNAs are loaded with the appropriate amino acid. We also know that the anticodon of the tRNA reads the codon sequence
of the mRNA, which in turn determines the sequence of amino acids linked together during protein synthesis. If the many components that
participate in translation (mRNA, aminoacyl-tRNAs) had to depend on random collisions in solution, the frequency would be so low that amino
acid polymerization would be very inefficient. The efficiency of translation is greatly increased by a remarkable macromolecular machine made up
of RNA and protein, called the ribosome. The ribosome is able to catalyze the polymerization of a protein chain at the rate of up to five amino
acids per second. Small proteins of 100 – 200 amino acids are therefore made in a minute or less.
A functional ribosome consists of two major subunits (large and small), each of which is composed of ribosomal RNA (rRNA) molecules and
dozens of proteins. Although a functional ribosome contains more than 80 proteins, this only accounts for a third of the ribosome by mass, with the
rest consisting of four different rRNAs. When not directly participating in translation, the two ribosomal subunits exist as separate entities in the
cytosol. They only come together when translation is initiated (usually close to the 5’ end of the mRNA). The small subunit facilitates base
pairing between the codon and anticodon sequences while the large subunit catalyzes the formation of peptide bonds between amino acids.
Each ribosome contains three sites that are able to bind tRNA in conjunction with the mRNA. The designation of each site is based on the type of
tRNA that it associates with. The A-site binds to the incoming aminoacyl tRNA and is the initial site of interaction between the ribosome and the
charged tRNA. The P-site binds to the peptidyl tRNA which is bonded to the growing polypeptide chain. The E-site binds to the exiting tRNA that
is no longer bonded to the polypeptide chain. Next, we will see how the three sites each contribute to the translation cycle required to add an
amino acid to the carboxyl-terminus of the growing polypeptide chain.
The Protein Translation Cycle
Step 1:
Incoming amino acid + tRNA is selected based on
anticodon to codon base pairing at the A-site
Step 2:
The bond between the end of the amino acid chain
and the tRNA at the P-site is broken
The free end of the amino acid chain then bonds to
the amino acid of the tRNA in the A-site
The ribosome shifts down the mRNA by three
nucleotides, placing the tRNAs in the E- and P-sites
Step 3:
The spent tRNA is ejected and the ribosome is reset
to bind another amino acid + tRNA at the A-site
The addition of each successive amino acid to a growing polypeptide chain requires a series of steps that involve the ribosome, mRNA, tRNA, and
a number of regulatory proteins. For the sake of simplicity we will first consider the translation cycle for the addition of an amino acid after the
process has already been initiated and the first three amino acids of the protein are already bonded together. We will also not consider the role of
the regulatory proteins. We will discuss the process of translation initiation and the critical role of the regulatory proteins later on.
Once translation has been initiated, each amino acid is added to the growing chain in a cycle of reactions consisting of three major steps. Here we
join the process with the P-site of the ribosome already containing a tRNA bonded to the end of the growing polypeptide of three amino acids. In
step 1, a tRNA carrying the next amino acid (#4) in the chain binds to the A-site by base pairing with the mRNA codon positioned there. Note that
the adjacent P-site and the A-site both contain bound tRNAs. In step 2, the carboxyl end of the polypeptide chain is released from the tRNA at the
P-site by breakage of the bond between the tRNA and its amino acid (#3). The polypeptide chain is then joined to the free amino group of the
amino acid linked to the tRNA at the A-site, forming a new peptide bond between #3 and #4. This is the central reaction of translation and is
catalyzed by the large ribosomal subunit. A subsequent conformational change in the ribosome shifts the two tRNAs into the E- and P-sites of the
large subunit. In step 3, another conformational change moves the mRNA exactly three nucleotides through the ribosome, and the tRNA in the Esite is released. This effectively resets the ribosome so it is ready to receive the next amino acyl tRNA in the now vacant A-site and Step 1 is then
repeated with a new incoming aminoacyl tRNA.
The Protein Translation Cycle
The mRNA is moved through the ribosome
by three nucleotides or exactly one codon
The Protein Translation Cycle
This three-step translation cycle is repeated each time an amino acid is added to the growing polypeptide chain. The amino group of the incoming
amino acid attacks the linkage between the peptidyl-tRNA and the carboxyl-terminus of the polypeptide chain. Therefore, each amino acid
addition involves the formation of a peptide bond between the carboxyl group at the end of the polypeptide chain and the free amino group of the
incoming amino acid. This also means that the polypeptide chain grows from its amino to its carboxyl end until a stop codon is encountered.
The formation of each peptide bond is energetically favorable because the growing C-terminus has been activated by the high-energy covalent
linkage to a tRNA molecule. This so called peptidyl-tRNA linkage activates the growing end and is broken in the course of each addition, but
immediately replaced by an identical linkage on the last added amino acid. Thus, the covalent linkage between the end of the polypeptide chain
and the tRNA is regenerated during each addition. In this diagram the amino acid side chains have been abbreviated as R1, R2, R3, and R4. All of
the atoms in the second amino acid in the polypeptide chain are shaded gray as a reference point.
Elongation factors enhance translation accuracy
EF-Tu
tRNA
Bacterial EF-Tu (Eukaryotic EF-1a)
The incoming amino-acyl tRNA is
bound to EF-TuGTP
- Peptide bond formation is blocked
Codon-anticodon pairing induces
GTP hydrolysis to EF-TuGDP
-Peptide bond formation is allowed
Rate of GTP hydrolysis is faster for
correct codon-anticodon pairs
We have previously seen that the accuracy of protein translation is enhanced by the proof reading activity of aminoacyl-tRNA synthetase enzymes.
However, ensuring that the correct amino acid is conjugated to the appropriate tRNA does not improve the accuracy of loading the correct
aminoacyl-tRNA into the A-site of the ribosome. In fact, the loading of an incorrect aminoacyl-tRNA into the A-site could result in the addition of
the wrong amino acid to the growing polypeptide chain. To reduce the likelihood of such an error in translation, two regulatory proteins or
elongation factors participate in the translation cycle. In bacteria, these elongation factors are known as EF-Tu and EF-G, while in eukaryotes the
homologous proteins are known as EF-1a and EF-2 respectively. For the sake of simplicity we will focus on the bacterial elongation factors.
Both EF-Tu and EF-G are GTPase enzymes that bind to GTP and cleave it to GDP and inorganic phosphate. This means that they can exist in two
forms distinguished by whether GTP or GDP is bound. Furthermore, the 3-dimensional conformation of each elongation factor depends on
whether GTP or GDP is bound, which means that GTP hydrolysis induces a significant change in the shape of the protein. As we will soon see, this
switch in conformation plays an essential role in the activity of each elongation factor.
EF-TuGTP associates with the aminoacyl-tRNA in the cytosol and protects the linkage between the amino acid and the tRNA from hydrolysis. This
also means that the aminoacyl-tRNA cannot participate in peptide bond formation while EF-TuGTP is bound. When the aminoacyl-tRNA plus EFTuGTP associates with the A-site, the appropriate codon to anticodon base pairing accelerates the rate of GTP hydrolysis by EF-Tu. Conversion of
EF-TuGTP to EF-TuGDP releases the aminoacyl-tRNA for participation in peptide bond formation. The rate of GTP hydrolysis by EF-Tu is
significantly slower in the presence of incorrect codon-anticodon base pairing, which in turn delays the release of the aminoacyl-tRNA for peptide
bond formation. During the resulting delay, the incorrectly base paired aminoacyl-tRNA is not able to attack the carboxyl terminus of the
polypeptide chain bound to the peptidyl-tRNA and is therefore more likely to leave the A-site.
Before translation can proceed, EF-TuGDP must leave the ribosome, thereby allowing the aminoacyl-tRNA to fully occupy the A-site. This
introduces another delay in the proceedings during which time an incorrectly base paired aminoacyl-tRNA that survived the first delay has another
opportunity to leave the ribosome. The combination of the two time delays caused by EF-Tu’s activity reduces the error rate to about one incorrect
amino acid per 10,000 couplings or an accuracy rate of 99.99%.
Elongation factors enhance translation accuracy
EF-Tu
Bacterial EF-Tu (Eukaryotic EF-1a)
The incoming amino-acyl tRNA is
bound to EF-TuGTP
- Peptide bond formation is blocked
1st delay
Codon-anticodon pairing induces
GTP hydrolysis to EF-TuGDP
-Peptide bond formation is allowed
tRNA
Rate of GTP hydrolysis is faster for
correct codon-anticodon pairs
EF-G
2nd delay
Bacterial EF-G (Eukaryotic EF-2)
EF-GGTP binds near the A-site
GTP hydrolysis to EF-GGDP
promotes the shift of the tRNAs into
the E and P sites
EF-Tu causes two time delays
Activity of EF-Tu makes translation
99.99% accurate
If you compare the 3-dimensional structures of EF-G, and ET-Tu bound to a tRNA, you will notice that they are quite similar. This similarity
indicates a shared mechanism for associating with the ribosome and for changing shape upon GTP hydrolysis.
EF-G does not directly affect the accuracy of amino acid addition to the growing polypeptide chain, but is essential for the subsequent translocation
of the mRNA through the ribosome. After EF-TuGDP leaves the ribosome, EF-GGTP binds to a region of the ribosome close to the A-site. This
binding event stimulates the GTPase activity of EF-G, which cleaves its bound GTP to GDP and inorganic phosphate. The resulting
conformational change to the EF-GGDP form drives the movement of the newly formed peptidyl-tRNA into the P-site and shifts the mRNA through
the ribosome by three nucleotides (or a single codon). The EF-GGDP is then released, with the ribosome having been reset to receive the next
aminoacyl-tRNA in the vacant A-site.
In summary, ET-Tu plays an important role in enhancing translational accuracy while EF-G promotes the translocation of the mRNA through the
ribosome. Both activities are crucial for the translation cycle, such that for every amino acid added to a growing polypeptide chain, one EF-TuGTP
and one EF-GGTP undergoes GTP hydrolysis.
Molecular animation showing the protein translation cycle based on the known 3-dimensional structures of the ribosome, tRNA, EF-Tu and EF-G.
Break Out
Proteins are synthesized in cytoplasmic extracts that
contain ribosomes and the appropriate mRNA.
The endoplasmic reticulum (ER) can be gently dispersed
into closed membrane sacs called microsomes.
A
B
Experiment:
Sample A: mRNA encoding a secreted protein fused
with GFP is added to a cytoplasmic extract without
membranes. After one hour, ER microsomes are added
to the extract, which is then incubated for another hour.
Sample B: Experiment is repeated with the ER
microsomes added at the same time as the mRNA and
incubated for one hour total.
Both samples are inspected for GFP fluorescence.
(A) The GFP portion prevents the fusion protein
from crossing the ER membrane
(B) Secreted proteins cross the ER membrane
while translation is occurring
(C) Secreted proteins cross the ER membrane
after translation
Break Out followup:
Cytoplasmic extracts can be made which contain ribosomes, tRNA, ATP, GTP, and all the requisite amino acids. With the addition of purified
mRNA, these extracts will support protein translation in vitro.
When cells are broken up or gently homogenized, the endoplasmic reticulum disperses into small closed membrane sacs called ER microsomes.
The ER microsomes mantain the orientation of the membrane in terms of the luminal versus cytoplasmic faces.
In this experiment, the above two procedures are combined to assess the targeting of a secreted protein to the lumen of the ER. You synthesize
mRNA encoding a large protein that is normally secreted from the cell fused with green fluorescent protein (GFP). The GFP fusion protein takes
about twenty minutes to be translated and is detectable in the microscope based on GFP fluorescenece. In Sample A, fusion protein mRNA is
added to a cytoplasmic extract without any membrane components. After one hour, ER microsomes are added to the extract, which is then
incubated for another hour. In Sample B, the same experiment is repeated except the mRNA and ER microsomes are added to the cytoplasmic
extract at the same time and incubated for one hour total. In the microscope we can see that Sample A has diffuse GFP fluorescence in the extract
with no signal in the microsomes. In contrast, Sample B exhibits significant fluorescence in the microsomes and none in the extract.
So what of the three possible interpretations?
Option A is not likely since in Sample B the GFP fusion protein clearly crosses the membrane of the ER microsome to accumulate in the lumen.
Option B is a reasonable interpretation since in Sample B the presence of the ER microsomes while translation is occurring correlates with
transport into the lumen. In Sample A, proteins that have already been translated appear to be incapable of crossing into ER microsomes added
later.
Option C is not likely since Sample A does exhibit GFP fluorescence in the extract indicating that translation has occurred, but no signal is present
in the microsomes. Furthermore, in both samples the same amount of time is spent in the presence of microsomes, so if transport occurred in
Sample B, there was enough time for it to occur in Sample A by a post-translational mechanism.
Co-translational transport
of secreted proteins
A
B
If we take a closer look at what occurred in each of the samples, we see that in Sample A complete fusion proteins were successfully translated by
free ribosomes in the extract, but these intact proteins were not able to cross the ER membrane post-translationally. Furthermore, since this is a
secreted protein that is meant to enter the ER lumen, the appropriate N-terminal signal sequence is present but still fails to support transfer across
the ER membrane after complete translation has occurred.
In Sample B, translation begins on free ribosomes in the extract, which are then targeted to the ER membrane (via the signal sequence), where
translation continues together with simultaneous transport across the membrane. This is the process of co-translational transport where the
ribosome is docked with a receptor channel that allows the newly synthesized polypeptide chain to enter the ER lumen as it is being made.
In both diagrams you will note that multiple ribosomes are translating the same mRNA simultaneously. This structure is called a polyribosome and
results from the fact that after one ribosome has initiated translation and moved down the mRNA, another ribosome can initiate while the first is
still translating. This ultimately results in several ribosomes moving down the mRNA and translating the same protein in a staggered row.
Co-translational transport
of secreted proteins
A
B
In the cell, translating ribosomes can exist free in the cytosol or associated with the ER membrane. Whether a ribosome falls into one category or
another entirely depends on the protein that it is synthesizing. In the case of cytoplasmic proteins, such as actin, there is no targeting sequence for
the ER membrane and the entire translation process occurs in the cytosol where the completed protein is released. If the mRNA encodes a secreted
protein or a transmembrane protein, then an ER signal sequence is present which targets the translating ribosome to the ER membrane -- resulting
in a membrane-attached ribosome. Membrane-attached ribosomes are what give the rough ER its unique appearance. Ribosomal subunits (small
and large) are recycled after each translation event and depending on which mRNA they happen to initiate translation on, will become either free or
membrane-attached. Thus, the two categories of ribsome are functionally equivalent and draw from a common pool of ribosomal subunits in the
cytosol.
Translation initiation and the Start Codon
In eukaryotes
Multiple initiation factors act at several steps
The small ribosomal subunit binds to a
special initiator tRNA + methionine
Requires the assistance of initiation factors
The small subunit recognizes the 5’ cap of
the mRNA + other initiation factors
The small subunit moves 5’ to 3’ on the
mRNA until it encounters an AUG codon
•Binds the initiator tRNA at the P-site
•Some initiation factors dissociate
•The large ribosomal subunit binds
So far we have considered the steps of the translation cycle in terms of how an amino acid is added to the C-terminus of an already growing
polypeptide chain. Now we will see how the entire process becomes started; in other words how translation is initiated. Since we have already
discussed the processing of eukaryotic RNAs (capping, polyadenylation, and splicing) we will continue with a focus on the initiation of eukaryotic
translation which utilizes two of these RNA modifications. But before we tackle how translation is initiated, it should be noted that most proteins
are synthesized with methionine as the first amino acid. This does not mean that all mature proteins contain this first methionine since many lose
their initial N-terminal sequences due to later proteolytic processing.
Initiation of translation requires multiple initiation factors (IFs) that act at several steps of the process. In the first step of the process, IFs promote
binding of the small ribosomal subunit (SSU) to a specialized initiator tRNA carrying methionine. The initiator tRNA is only used to carry the
methionine that forms the first amino acid of most proteins. This is the only case in which a tRNA associates with a ribosomal subunit in the
absence of mRNA. When methionine is incorporated elsewhere in a protein, it is delivered to the ribosome by a different set of standard tRNAs.
IFs also associate with the mRNA at both the 5’-cap and the poly-A tail. These two covalent modifications define the intact ends of the mRNA and
through their interactions with IFs ensure that only intact mRNAs are subject to translation. The small ribosomal subunit with its associated
initiator tRNA+methionine recognizes the mRNA and the IFs bound to the 5’-cap. This recognition event positions the small ribosomal subunit at
the 5’ end of the intact mRNA and upstream of the translational start site. The SSU then uses the energy of ATP hydrolysis to move from 5’ to 3’
down the mRNA until it encounters the first AUG codon. The initiator tRNA base pairs with the AUG codon and several IFs dissociate from the
complex. The remodeling of the initiation complex allows the large ribosomal subunit to bind, and the initiator tRNA+methionine is loaded into
the complete P-site. This assembly process produces a functional ribosome with a tRNA loaded in the P-site and the A-site open to an incoming
aminoacyl-tRNA.
Translation initiation and the Start Codon
The complete ribosome can now begin translation
The next tRNA + amino acid binds at the A-site
Once the complete ribosome is assembled with the initiator tRNA+methionine loaded at the P-site, the A-site receives the aminoacyl-tRNA
carrying the second amino acid encoded by the mRNA. The translation cycle then proceeds with the formation of the first peptide bond between
the methionine and the second amino acid in the polypeptide chain.
In most cases, translation will begin with the first AUG that is encountered by the small ribosomal subunit as it moves down the mRNA. In
eukaryotes there are sequences around the AUG that facilitate the recognition process. For a few genes, the first AUG of the mRNA may not be
surrounded by these additional recognition sequences, in which case the SSU may skip the first AUG and initiate translation at another AUG. In
these rare cases, proteins with different N-termini can be produced from the same mRNA.
Translation termination and the Stop Codon
Three codons signal the stop of translation:
UAA
UAG
Stop codons
UGA
Release factor binds to stop codons in the A-site
Release factor does not carry another amino acid to
bond to the growing amino acid chain
When we discussed the genetic code we noted that three codons are defined as stop codons. These stop codons (UAA, UAG, UGA) signal the end
of translation for a particular mRNA, and are decoded in the same reading frame as the growing polypeptide chain. There are no tRNAs with the
appropriate anticodon sequence to base pair with any of the three possible stop codons. Therefore, when a ribosome encounters a stop codon in the
A-site, instead of binding to an aminoacyl-tRNA, the codon binds to a protein release factor. The release factor resembles tRNA in 3-dimensional
shape, which facilitates it binding in the A-site. However, the release factor does not carry an amino acid for addition to the growing chain.
Translation termination and the Stop Codon
Instead of being bonded to the next amino acid,
the end of the amino acid chain bonds to an -OH
group from water (another example of hydrolysis)
Releases the amino acid chain from the ribosome
The ribosome complex disassembles
The association of a release factor in the A-site induces the ribosome to catalyze the addition of H2O to the carboxyl end of the amino acid chain
(hydrolysis reaction). The polypeptide is therefore released from the ribosome since the tRNA linkage is not regenerated. This underscores the
fact that the polypeptide chain is held to the ribosome primarily via its linkage to the peptidyl-tRNA. The ribosome then disassembles into separate
small and large subunits which are recycled for use in other translation events.
HIV Protease
3-dimensional structure of the HIV protease enzyme. Note that the active protease exists as a dimer of two subunits (shaded green and orange
respectively).