BIO353.

01 2021/22-1

Lecture 11: Translation

Slide 2
Translation described the process of synthesizing proteins from information encoded by mRNAs. As we
have seen in the previous Lecture, multiple alternative mRNAs can be generated from the same gene,
which may result in alternative protein isoforms with altered function, specificity, or enzymatic activity.

Slide 3
Protein synthesis requires a few basic ingredients and molecular machines. The instruction (the
cookbook if you want) is the processed (i.e. capped, polyadenylated, and spliced) mRNA. Ribosomes
(the food processor) utilize this information to catalyze the synthesis of a single continuous polypeptide
chain using the 20 proteinogenic amino acids as elementary building blocks (ingredients). Charged
tRNAs (i.e. tRNAs loaded with a specific amino acid) form an intermediate that recognizes appropriate
codons in the mRNA and presents the amino acid with the right geometry during the synthesis reaction
(= peptidyltransferase reaction).

Slide 4
DNA consists of 4 different bases (A, C,T, and G), which encode which of the 20 amino acids will be
inserted into the growing polypeptide next. This mandates that at least three consecutive bases (i.e.
triplets) within the mRNA are required to specify an amino acid uniquely. As you can see, a two-letter
word (e.g. AC, CT, TT, CA, …) would not be sufficient since only 16 different combinations are possible
2 3
(4 = 16). On the other hand, the codon triplets provide more possibilities than necessary (4 = 64
possible combinations). Thus, different codon triplets may code for the same amino acid, which results
in the degeneration of the genetic code. Most amino acids are specified by two alternative codons, while
others can be encoded by six different base triplets.

Slide 5
In addition to instructing the insertoion of a specific amino acid into the polypeptide, some codons also
have specific function. For instance, the codons UAA, UAG, and UGA specify the end of the open
reading frame (= STOP codon) and will result in termination of translation when encountered. The codon
AUG is a universal START codons but also instructs insertion of a methionine when encountered in the
middle of the mRNA in addition to the beginning of the protein. In addition, UUG, AUU, and GUG can
serve as alternative start codons in prokaryotes.

Slide 6
The genetic code only works if it is read in the right frame. Since amino acids are specified by three
consecutive bases in the mRNA, theoretically three alternative frames are possible. Thus, recognition
of the correct frame is an important aspect of translation initiation. The frame is decided by the first
codon that is translated and mechanisms exist to increase the specificity of this process. The results are
obvious, frame shifts may result in synthesizing a useless polypeptide. Often, however, the alternative
frames are riddled with STOP codons, which would prevent accumulation of malfunctioning or
functionless short peptides.

Slide 7
The mechanisms by which the START codon, and ultimately the frame, is identified, differ between pro-
and eukaryotes. Prokaryotic mRNAs contain ‘ribosome binding sites’ that are located upstream of the
translated sequence, typically between 3 to 9 bp upstream of the START codon. As the name implies,
these sites recruit the ribosome and position the START codon into a specific pocket inside the
ribosome. Eukaryotic mRNAs typically specify the synthesis of only a single protein. Prokaryotic
mRNAs, however, often instruct the simultaneous translation of multiple proteins from the same
messenger. These prokaryotic mRNAs contain multiple open reading frames (also called cistrons),
which each are preceded by a ribosome binding site. Thus, multiple ribosomes bind independently to
different translation starts on the same mRNA.

Slide 8
Similar as to what we have seen for promoter sequences, the comparison of multiple sequences around
the translation start of different genes identifies a consensus sequence for the ribosome binding site.
This sequence is also known as the Shine-Dalgarno sequence.
Slides 9 / 10
The Shine-Dalgarno sequence is recognized by an RNA component of the small ribosomal subunit. The
16S RNA contains a sequence that is complementary to the ribosome binding site and base pair
interactions between the 16S RNA and the mRNA position the ribosome. Other interactions that take
place at the START codon also contribute to the specificity of the match. You can see that none of the
translation starts shown here is a perfect complementary match to the 16S RNA. Similar to promoter
sequences, some mRNAs make stronger interactions and are translated more efficiently than mRNAs
that have a weak similarity with the consensus sequence.

Slide 11
The process of initiation of translation is very different between pro- and eukaryotic cells. In prokaryotes,
the ribosome can be recruited to any position in the middle of the mRNA through the interactions we
just discussed. In eukaryotes, the ribosome is always recruited to the 5’-CAP of the mRNA and scans
along the mRNA molecule until it encounters a START codon. Eukaryotic START codons also are
flanked by sequence motifs that allow the ribosome to distinguish between a AUG codon at the
beginning and a AUG codon in the middle of the reading frame. These sequence motifs are referred to
as the Kozak sequence (after Emily Kozak, who first described their existence).

Slide 12
Important molecules during the process of translation are the transfer-RNAs (tRNAs), which recognize
the codon triplets through complementary anticodons and carry the amino acids. The conceptual
structure is often described as a clover leaf (= yonca) but the actual three-dimensional folding of tRNAs
is more complex. Base pair interactions within the tRNA define various loops of the overall L-shaped
molecule. The anticodon loop is located at the tip of the molecule, whereas the amino acid is attached
to the 3’-end of the tRNA opposite the anticodon loop. The 3’-strands of the acceptor arms of all tRNAs
end on the same CCA sequence, which is not encoded by tRNA genes but added posttranscriptionally
by a CCA-adding enzyme. This sequence motif is important for the attachment of amino acids to tRNAs.

Slide 13
tRNAs contain a large number of unusual nucleotides/bases for different reasons. For instance,
pseudouridine bases contribute to structural stability, while dihydrouridine makes the tRNA molecules
more resistant to degradation by RNases. Many tRNAs also contain an inosine base in the anticodon
loop, which is part of the triplet that interacts with codons in the mRNA. As we will see, inosine can
interact with multiple bases and contributes to the ability of tRNAs to recognize more than just one
defined codon triplet. Since transcription of tRNAs from their respective genes can only add A, U, C,
and G nucleotides, all of the base changes are made posttranscriptionally through deamination or other
chemical modifications of the existing bases through RNA editing.

Slide 14
The tRNAs are the agents that read the genetic triplet code. Thus, it is very important that the correct
amino acids are loaded onto the right tRNA. If tRNAs would carry the wrong amino acid, the protein
sequence would be changed, similar to the effect of a mutation in the gene itself. Therefore, the pairing
of amino acids with tRNAs is sometimes likened to a second genetic code.

Slide 15
The addition of an amino acid to the 3’-end of a tRNA is referred to as the ‘charging’ of tRNAs. The
reaction is synthesized by tRNA synthetases. Different tRNA synthetases exist for different amino acids
but the same synthethase may attach the same amino acid to different tRNAs with different codons.
Thus, tRNA synthetases are the molecules that determine the second genetic code and their specificity
is critical for the survival of cells and the whole organism.

Slide 16 / 17
The charging reaction is a two-step process. First, a high energy bond is transferred to the amino acid
in an adenylylation reaction (yes, you read correctly). An adenosine monophosphate (the adenyl) is
added to the carboxyl group of the amino acid. In a second step, the amino acid is transferred to the
tRNA under preservation of the high energy bond.

Slides 18 / 19
There are only 20 tRNA synthetases (also called aminoacyl-tRNA synthetases), one for each amino
acid, but the number of tRNAs is much higher. Thus, the same tRNA synthetase must recognize different
isoaccepting tRNAs to account for the degeneration of the genetic code. Some degenerate codons are
recognized by the same tRNA through ‘wobbling’ (will be discussed later) but as you can see, some
amino acids are also specified by very different codons that cannot be recognized by the same tRNA.
For some amino acids up to five different isoacceptor tRNAs exist.

Slides 20 / 21
Aminoacyl-tRNA synthetases must be able to recognize specific groups of isoaccepting tRNAs. A simple
assumption is that the enzymes would recognize the anticodon because that is the most unique part of
every tRNA. However, this is not always the case. Additional sequence motifs, including a critical
discriminator base close to the 3’- end of the tRNA contribute to the specificity.

Slide 22
An additional complication is the fact that amino acids are rather small molecules and some of them
have a very related structure, which makes it difficult to discriminate them from each other.

Slide 23
The tRNA synthetases need to account for that. While it is easy to discriminate between tyrosine and
phenylalanine due to the presence of the hydroxyl group in tyrosine, discriminating between unipolar
amino acids, such as valine and isoleucine, is much trickier. Size exclusion in an editing pocket may
help here. Thus, both valine and isoleucine are substrates for the initial charging reaction but the larger
isoleucine cannot enter an editing pocket that would remove the charged amino acid. The smaller valine,
however, is removed.

Slide 24
Ribosomes are macromolecular complexes that consist of two subunits. Ribosomal subunits are often
referred to by their sedimentation coefficients, which are measured in Svedberg (S). The unit describes
the speed of migration in µm/s of particles in a centrifugal field of 1 million gravities. Thus, a particle with
a coefficient of 30S will move 30 µm/s in the centrifuge tube. Larger particles tend to migrate faster but
the speed depends on other parameters, such as shape of the particle, and the unit is non-linear. The
smaller subunit has a coefficient of 30S, the larger of 50S, and the combined full ribosome of 70S (in
bacteria)

Slide 25
Ribosomes are ribonucleoproteins, meaning that they consist of RNA and proteins. The isolated
subunits can be analyzed for their protein content by 2D gel electrophoresis, which separates proteins
by mass and charge. The 30S subunit of E. coli contains 21 proteins, which are termed S1 – S21 (S =
small) and the large subunit consists of 34 proteins (L1 – L34; L = large).

Slide 26
The small subunit contains a single large RNA in addition to the 21 proteins, the 16S rRNA in E. coli.
The large subunit contains the short 5S and the long 23S rRNAs. It may be confusing but in this case
the S refers to the sedimentation coefficient in Svedberg again. Ribosomal RNAs form complex but
defined secondary structures through intramolecular base pairing as shown on the left.

Slide 27
The small and large ribosomal subunits contain defined sites that participate in polypeptide synthesis.
The small subunit includes the ‘decoding center’, where interactions between the mRNA and the tRNAs
take place to read and where the codons of the mRNA are read. The large subunit catalyzes the
synthesis reaction in a defined peptidyl transferase center.

Slide 28
Eukaryotic ribosomes are structurally very similar to prokaryotic ones but include more proteins and
RNAs. The small subunit is composed of 33 proteins in addition to the 18S rRNA, while the large subunit
is made out of 49 proteins and the 5S, 5.8S, and 28S rRNAs.

Slide 29
Ribosomes go through the ribosome cycle, which describes their binding to the mRNA, the recognition
of the START codon, the assembly of the active complex, the peptidyl-transferase reaction, the
termination of translation, and the recycling of the ribosome.
Slide 30
Multiple ribosomes can simultaneously engage with the same mRNA both in pro- and eukaryotes to
form polysomes (= polyribosomes). While mRNA can be used for translation as it emerges from the
RNApol in bacteria, the mRNA is transported out of the nucleus for translation in the cytoplasm.

Slide 31
The peptidyl transferase reaction that is catalyzed by the ribosome occurs between the growing
polypeptide chain and the next amino acid to be incorporated into the protein. Both reactants are
attached to tRNAs. The complex of the polypeptide and the tRNA is called the peptidyl-tRNA similar to
the charged tRNA, which is an aminoacyl-tRNA. The reaction takes place by an attack of the amino
group of the aminoacyl-tRNA onto the ester group of the peptidyl-tRNA. As you can see, this will break
the bond between the peptide and the tRNA, while the amino acid remains attached to its tRNA carrier.
As a consequence, the peptide is transferred to the tRNA that carries the next amino acid.

Slide 32
Both the decoding center and the peptidyl transferase center define three positions within the ribosome
that are designated as the E, P, and A sites. The names refer to the interactions that are made with
differently charged tRNAs during the synthesis reaction. The A site contains the aminoacyl-tRNA, the P
site the peptidyl-tRNA, while the Esite (for exit) will hold the uncharged tRNA from which the peptide
was transferred during the last reaction cycle. The peptide will be transferred from the P to the A site
and then the whole assembly moves one position forward during each repetition of the cycle.

Slide 33
This slide shows the molecular structure of the large and small ribosomal subunits with the three tRNAs
that occupy the A, P, and E sites attached. The tips of the tRNAs interact with the mRNA inside the
decoding center. They are positioned such that the 3’-accptor stems are in close proximity with each
other. In this illustration proteins are shown in darker colors and ribosomal RNAs in lighter colors. You
can appreciate that both the decoding and the peptidyl-transferase centers are mostly defined by
ribosomal RNAs and that proteins are largely absent from these sites. Evolutionary, RNA came before
DNA and the first proteins that were synthesized also were made by catalytic RNAs that became the
ribosome. The ribosomal proteins occurred later and further stabilized the complex and positioned the
catalytic RNAs so that they are in an optimal conformation.

Slide 34
A closer look onto the arrangement of the three tRNAs that occupy the A, P, and E sites. The 3’-stems
that are charged with the amino acids of the tRNAs in the A and P sites are in close proximity, which is
necessary for the peptidyl transferase reaction to occur.

Slide 35
The large ribosomal subunit also defines an exit tunnel for the polypeptide that is synthesized and which
emerges between the A and the P sites between which the peptidyl transferase reaction occurs.

Slide 36
Similar to replication and transcription, the process of translation can be broken down into three different
phases. The initiation phase is characterized by the initial binding of the ribosome with the mRNA and
the identification of the translation START codon. Polypeptide synthesis occurs during an elongation
phase and terminates when a STOP codon is encountered. The ribosome needs to be cleared from
factors that are stuck inside the ribosome at the end of translation. All three phases are controlled /
supported by specific regulatory factors.

Slides 37 / 38
Initiation is achieved differently in pro- and eukaryotes but some of the factors are related. The START
codon of an mRNA is usually not directly at the beginning of the messenger but several nucleotides
away from its 5’- end. We have seen earlier that the START codon is preceded by ribosome binding
sites (= Shine-Dalgarno sequences) that interact with a specific stretch of the 16S rRNA of the small
subunit. A specific initiator tRNA, which is charged with a formylated methionine (fMet) takes part in the
initiation process. The mRNA interacts with the initiator tRNA and the small ribosomal subunit such that
the AUG START codon is positioned in the P site and base paired with the fMet-tRNA. Once this
preinitiation complex has formed, the large ribosomal subunit is recruited.
Slides 39 / 40
The start codon typically is an AUG codon (exceptions exist, especially in prokaryotes), which is
translated into the amino acid methionine when encountered in the middle of the mRNA coding
sequence. Why does the initiator tRNA for the first AUG at the transcription start carry a N-formyl
methionine? Formylated methionine shares structural similarity with the synthesized polypeptide chain
beyond the ester group that connects the amino acid to the tRNA and is similar to the peptide bond
between the last and second-to-last amino acid. This may give the fMet-tRNA a more stable geometric
positioning inside the forming preinitiation complex. Eukaryotes do not require a formylated methionine
during initiation.

Slide 41
At the end of the ribosome cycle, the large and the small subunit are still attached to each other even
though the ribosome is not occupied by tRNAs anymore. A specific initiation factor, IF3, binds to the
small ribosomal subunit and forces the dissociation from the large subunit. IF3 binds directly to the E
site so that no tRNA could occupy this site during initiation. This forces the fMet-tRNA to enter either the
P or the A site.

Slide 42
Occupation of the A site is also prevented by initiation factors. A complex of IF1 and IF 2 binds to the A
site, which positions the fMet-tRNA in the P site. IF2 directly interacts with the fMet-tRNA to attract it to
the forming complex and is loaded with a GTP molecule, which will be released during binding of the
large subunit. The combination of mRNA, small ribosomal subunit, fMet-tRNA, and the three initiation
factors is called the 30S initiation complex.

Slide 43
Release of IF3 allows the large subunit to interact with initiation complex. The large subunit also contains
a specific site that is known as the factor binding center. This site is important for a variety of switches
that occur during translation. During initiation, it stimulates the hydrolysis of GTP by IF2, which results
in changed interactions between the initiation factors, the fMet-tRNA and the ribosomal subunit. As a
consequence, IF1 and 2 are released and the readily assembled ribosome (the 70S initiation complex
is now only occupied by the initiator tRNA and the mRNA. Next, the complex can start translation of the
next codon.

Slide 44
Initiation in eukaryotes requires additional factors, which, however, have similar effects on the small
ribosomal subunit. Binding of eIF3 (= eukaryotic initiation factor 3), along with other factors results in
dissociation of the subunits. eIF3 forms a complex with eIF1 and 5, which block the E site of the
ribosome. eIF1A occupies the A site and eIF2, which similar to prokaryotes binds GTP, positions the
initiator tRNA into the P site through interactions with eIF5. Even though the assembly is more complex,
essentially the same conditions have been achieved that we have seen before. The difference is that in
this 43S preinitiation complex, no mRNA is bound yet.

Slides 45 / 46
The mRNA needs additional preparation before it can be loaded onto the 43S preinitiation complex. The
cap-binding protein eIF4E binds to the very 5’-end of the mRNA and forms a complex with eIF4A, B,
and G. Some of these factors also have helicase activity, which breaks the secondary structure of
mRNA. The mRNA that is decorated with these factors can now be loaded onto the preinitiation complex.

Slide 47
We have already discussed this but here it is shown as a reminder. The eIF4 complex on the 5’-end of
the mRNA interacts with polyA-binding proteins to bend the mRNA into a circular shape, which allows
ribosomes that reach the end of the messenger to reinitiate efficiently at the 5’-cap.

Slide 48
Another difference between pro- and eukaryotes is that the eukaryotic initiation complex does not form
at the START codon but at the 5’- end of the mRNA. The START codon itself can be several hundred
nucleotides away from the sites at which the complex forms. Thus, the complex slides along the mRNA
until it encounters an ATG codon through a scanning process. The interaction of the loaded initiator
tRNA with the AUG and the presence of a Kozak sequence flanking this site allow for proper recognition.
Slides 49 / 50
When the AUG is encountered, most, but not all, initiation factors leave the complex, which allows the
large ribosomal subunit to bind. Paradoxically, a new factor eIF5B is recruited during this process. As
for prokaryotes, the interaction of the factor binding center with eIF5B triggers GTP hydrolysis and
release of the remaining factors, which makes the ribosome ready for elongation.

Slide 51
Although ribosome assembly begins always at the 5’-end of the mRNA followed by scanning, some viral
mRNAs can recruit a ribosome to sequences in the middle of the molecule. The sites that can achieve
this are generally known as internal ribosome entry sites (IRESs). The recruitment of the ribosome to
internal AUG codons can be achieved in different ways. Some IRES sequences form complex
secondary structures that resemble the 5’-end of an mRNA and bind eIF4G, which in turn recruits the
43S preinititation complex. Others form even more sophisticated structures that resemble a tRNA that
can occupy the E site of the ribosome, which will then go into elongation. In this case, the first codon
does not have to be an AUG codon.

Slide 52
During elongation, a new aminoacyl-tRNA with specificity for the next codon is recruited to the A site
and a new peptide bond is formed by transferring the formyl-Methionine (Methionine) from the P site to
the A site amino acid/tRNA. The complex then moves one codon further, which brings the peptidyl-tRNA
into the P site and the empty tRNA into the E site, where it will be evicted during the next cycle step.

Slide 53
In principle, any of the multiple tRNAs could enter the A site but a specific elongation factor, EF-Tu,
ensures that only aminoacyl-tRNAs that make proper base pair interactions with the A site codon can
participate in the peptidyl transferase reaction. EF-Tu binds strongly to tRNAs when loaded with GTP.
If, and only if, the tRNA can form proper base pairs with the codon, will EF-Tu be positioned in the factor-
binding center. This interaction triggers GTP hydrolysis and the release of EF-Tu from the tRNA. As a
consequence, the aminoacyl-tRNA is now positioned in the A site.

Slide 54
EF-Tu requires the cofactor EF-Ts, which functions as a guanylyl exchange factor and replaces the
GDP with GTP for a new round of the loading cycle.

Slide 55
Thus, EF-Tu contributes largely to the accuracy of translation. Wrong tRNAs that do not match the codon
may enter the A site but will not be released from EF-Tu because it does not interact with the factor
binding center in any meaningful way. Additional specificity is provided by the 16S rRNA of the small
subunit, which makes electrical interactions with properly paired bases but not with base mismatches.

Slide 56
A third proofreading mechanism is provided by the fact that the aminoacyl-tRNA has to rotate inside the
A site to be positioned properly for the peptidyl transferase reaction. The rotation is referred to as
accommodation. Since it puts a lot of strain on the base pairs between the anticodon and the codon,
mismatched tRNAs dissociate more readily during the process.

Slide 57
Another dramatic conformational change occurs during translocation, i.e. the movement of the peptidyl-
tRNS form the A to the P site and of the uncharged tRNA from the A to the E site. The small ribosomal
subunit rotates relative to the large one to open the passage between sites, which are isolated by
barriers in the classic state.

Slide 58
How does the ribosome move forward along the mRNA? Translocation requires another factor that
serves as the motor of the movement. EF-G can enter the A site in a GTP-bound state in addition to the
peptidyl-tRNA that is present in this site after the last transferase reaction. Again, EF-G interacts with
the factor binding center in the large subunit, which triggers GTP hydrolysis. The reaction triggers a
conformational change in EF-G, which extends inside the A site and pushes the peptidyl-tRNA, as well
as the two uncharged t-RNAs in the P and E site one position forward. The E site tRNA leaves the
ribosome, while the P site tRNA moves to the E site.
Slide 59
EF-G is an amazing protein that in the GDP-bound elongated form shares overall structural similarity
with an aminoacyl-tRNA bound to EF-Tu.

Slide 60
Termination of translation requires special release factors that read the STOP codon. Surprisingly, these
release factors are proteins, not RNAs, which again adopt an overall shape that closely resembles a
tRNA.

Slide 61
The release factor forms a peptide anticodon that detects the STOP codon.

Slide 62
The release factor RF1 enters the A site and presents a defined peptide sequence (GGQ) as potential
acceptor for the transferase reaction. However, this transfer is not successful and the peptide is
released. It also recruits an auxiliary factor, RF3, in a GDP-bound state, which helps the clearance of
RF1 from the ribosome. Exchange of GDP for GTP triggers a conformational change in RF3 that evicts
RF1. It can now enter the factor binding center, which results in GTP hydrolysis and eviction of RF3
from the ribosome, which is now left with two uncharged tRNAs in the P and E site.

Slide 63
It would be reasonable to assume that the ribosome simply dissociates after the peptide has been
released. Somehow, the disassembly and clearance of the ribosome is more complicated and requires
a ribosome recycling factor (RRF) that enters the A site and is pushed forward by EF-G similar to a
tRNA. This results in the eviction of the remaining two tRNAs, ahdich allows IF3 to bind to the vacated
E site, which keeps the large subunit from rebinding to the small subunit.

Slide 64
As you can now appreciate, translation is a complex and complicated process and eventually something
can go wrong, which requires attention by specific mechanisms to ensure proper response inside cells.

Slides 65 / 66
Eventually, the translated mRNA is broken and the ribosome does not encounter a STOP codon before
it reaches the end of the mRNA molecule. This would prevent ribosome recycling and the complex would
get stalled. Prokaryotes possess special tmRNAs (for tRNA/mRNA hybrids. These tmRNAs form
secondary structures at their 5’-end that mimic a tRNA that is charged with Alanine. The structure is
recognized by EF-Tu and can be loaded to the A site and serve as an acceptor for the peptide during
the next transfer reaction. The remainder of the tmRNA can now serve as a regular mRNA and codes
for a specific peptide sequence that marks the translated protein for degradation. The ribosome can
now continue until it reaches the STOP codon inside the tmRNA when it is disassembled by the regular
sequence of events.

Slide 67
In eukaryotes, the same is achieved by processes that are called ‘no go’ and ‘nonsense-mediated
decay’. If the ribosome is stalled, protein factors (Dom34/Hbs1 in mammals) are recruited that
disassemble the ribosome. The difference really is whether the ribosome stops in the middle of the
mRNA or at the very end, i.e. if it does not encounter a STOP codon before reaching the end of the
mRNA molecule.

Slide 68
The factors also recruit exonucleases (and other enzymes) that result in the degradation of the
broken/malfunctioning mRNA and the peptide that has been synthesized.

Slide 69
We have seen that spliced mRNAs are decorated with exon junction complexes. These complexes are
removed during the first round of translation by the ribosome.
Slide 70
This protects mRNAs that contain STOP codons in the middle of the sequence from being translated
into proteins. Typically, no introns occur after the STOP codon, thus if a ribosome encounters a STOP
ahead of remaining exon junction complexes the mechanism of ‘nonsense-mediated decay’ is triggered.
This includes the recruitment of several factors and enzymes that decap and deadenylate the mRNA,
which allows degradation of the faulty mRNA both from the 5’ and the 3’-end.

End of transcript !!! - (shf, 27 .11.2021)