BIO353.

01 2021/22-1

Lecture 12: The Genetic Code

You are all aware of the genetic code, which instructs how genetic information is read during protein
translation. At first glance, the genetic code appears random but there are actually interesting
properties build into the code that make it robust to mutations that may occur in the genome.

Slide 2
A short summary of the properties of the genetic code, which follows three simple rules. Amino acid
identity is coded in triplets of bases in DNA / RNA, it is non-overlapping in the sense that adjacent
triplets form separate information unites, and there are no gaps in the code. A larger exception to the
last rule is the fact that genes consist of exons and introns but as soon as splicing has occurred, the
code consists of a single uninterrupted open reading frame.

Slide 3
This is an illustration of the standard genetic code as you know it. The 20 proteinogenic amino acids
are encoded by different numbers of base triplets (between 1 for Methionine to 6 for Arginine, Leucine,
and Serine). Since multiple, not necessarily related codons instruct the translation of the same amino
acid, the code is referred to as being degenerate. Some codons have special meaning, such as the
start codon AUG or the three stop codons UAA, UAG, and UGA.

Slide 4
Obviously, mutations at DNA level change the meaning of the DNA/RNA sequence. Even single
nucleotide mutations can have a great impact on the protein that is translated from a gene/mRNA
sequence. Synonymous mutations are those that change the DNA/mRNA sequence but do not result
in a change of the encoded amino acid. In contrast, missense mutations result in a different amino
acid that is used during translation. Nonsense mutations are those that result in the change from a
codon that codes for an amino acid into one that is read as a stop codon. All of these mutations can
occur during DNA synthesis when individual bases are incorrectly replicated. We have also seen that
other mechanisms, such as polymerase slippage or DNA repair can give rise to insertions or deletions,
which often result in frameshift mutations. Even the insertion of a single nucleotide will change the
meaning of the triplet code after the base insertion and a completely unrelated, new protein is
synthesized.

Slide 5
The degeneration of the genetic code is somewhat variable. Some amino acids, such as Methionine
and Tryptophan are only encoded by a single codon, others are specified by 2, 3, 4, or 6 alternative
codons.

Slide 6
Because sometimes the different alternative codons are very different from each other in sequence,
different tRNAs exist that are charged with the same amino acid but present different anticodons.
Such tRNAs that are charged with the same amino acid are called ‘isoaccepting’ tRNAs.

Slide 7
The organization of the genetic code provides a certain degree of protection against some of the most-
common point mutations that may occur in the genome. As we have seen before, transitions are more
common than transversions. If you take a look at the codon table, you will see that transition in the first
base of the codon (i.e. U / C and A / G changes) will often specify amino acids with similar properties,
such as Serine / Proline changes for U/C transitions or Isoleucine/Valine changes for A/G transitions.
Those changes in the code correspond to transitions. The less frequent transversions, however, would
result in more dramatic amino acid changes.

Slide 8
This slide serves as a reminder of the most frequent mutations that can occur in the genome. We have
seen that spontaneous deamination is responsible for most base changes. Deamination of A, G, C,
and 5-methyl G all result in transition mutations.
Slide 9
Another frequent source of mutations are tautomeric shifts during replication. Again, the bases that
base pair with enol-T or imino-C would cause transition mutations but not transversions.

Slide 10
Changes in nucleotides can occur in any of the three codon positions. Changes that occur in the third
base are usually the most harmless ones. Often all four of the possible codons, regardless whether
the mutation is caused by a transition or transversion, specify the same amino acid (8 out of 20 amino
acids).

Slide 11
In those cases where the same amino acid is specified by two codons, they are typically identical for A
/ G or U / C, thus, again transition mutations would not change the encoded amino acid.

Slide 12
A similar observation can be made for the second base in the codon. Non-polar amino acids are
specified by codons that use U / C in the second base, whereas polar or charged amino acids use A
or G. Thus, even if a different amino acid is inserted into the polypeptide chain, often these amino
acids have related properties and eventually do not change the overall function of the protein.
Naturally, this is not always the case but may be beneficial for some genes.

Slide 13
This slide summarizes the correlation of nucleotides in the second base with the overall nature of the
amino acids that are specified.

Slide 14
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There are other regularities that can be observed in the code. If the 1 and 2 nucleotide are a C or G
(any combination), all four possible codons code for the same amino acid. If these bases are an A or a
U, it is usually only two codons that specify the same amino acid. This may be explained by the fact
that G and C form more stable base pairs than A / U, which relaxes the need for a third base to
uniquely determine the amino acids.

Slides 15 / 16
We have already seen that multiple isoaccepting tRNAs exist that read the different codons of the
degenerate genetic code. However, some tRNAs are able to read several related codons that differ in
the third position. This ability is based on the ‘wobble concept’, which was first proposed by Francis
st
Crick. The concept implies that in the third position of the codon (= 1 position or 5’end of the tRNA
anticodon) the geometric constraints are reduced and that unusual base pairs can form. For instance,
G nucleotides can also form unusual hydrogen bonds with U bases. This does not usually occur but
because of the specific structure of the anticodon loop is allowed for tRNA-mRNA interactions.
Moreover, inosine bases pair with any of the three bases C, U, and A in the first anticodon position.

Slide 17
rd
The 3’-base of the codon (= the 3 base) pairs with the first base at the 5’-end of the anticodon. This
base is located at position 34 at the tip of the anticodon loop. The tRNA structure takes a sharp loop in
the 5’ direction. In a perfect helical structure the bases are stacked in a planar arrangement with each
other. The 34 base lacks a stacking base at the 5’ side, which gives it more flexibility for wobbling, i.e.
forming unusual base pairs.

Slide 18
This diagram highlights some of the most common base modifications that are found in tRNAs. You
can see that base 34 and 37 are the most diverse. As shown on the previous slide, base 34 is the first
base of the anticodon, and inosine is common here, so are some other unusual bases that allow for
wobbling. Base 37, which is not part of the anticodon itself but 3’ of the anticodon also contains
unusual bases.

Slide 19
The modified bases in position 37 often are bulky structures that force base stacking with the last base
of the anticodon, which gives it less flexibility and, therefore, requires perfect base pairing in the codon
sequence.
Slide 20
The table gives an overview of the possible wobble bases if they are encountered in the first position
of the anticodon.

Slide 21
There are a total of 64 codons, 61 of which code for amino acids. Because of the wobble mechanism
only 31 aminoacyl-tRNAs are required to read all of these codons.

Slide 22
Interestingly, some organells and bacteria can do away with even a lower number of tRNAs. For
instance, eukaryotic mitochondria only use 22 tRNAs to accommodate all possible codons. The
anticodon sequence is given in brackets. You can see that all of them use an unmodified U base in the
first position. In these tRNAs, the U can essentially base pair with all 4 bases in the third position of
the codon, a property known as ‘superwobble’.

Slide 23
The genetic code is also often called the standard genetic code and it is assumed to be universal.
However, some codons can have different meaning in different organisms.

Slide 24
Individual tRNAs are often encoded by multiple genes. A reasonable assumption (with limitations) is
that the number of genes for tRNAs that read a specific codon is somehow linked to the
importance/abundance of this codon. Here you can see that some anticodons of isoaccepting tRNAs
are favored. Expression levels, however, can be different for different tRNA genes even if they include
the same anticodon. The number of transcripts for each species of tRNAs is indicated by the size of
the label (read frequency).

Slide 25
Even if multiple codons exist for a single amino acid, some codons are favored over others (e.g.
because of stability of the interaction. This is reflected in the codon usage table shown here. For
instance, the amino acid valine is encoded by four codons (GUU, GUC, GUA, and GUG) but GUG is
highly favored over the other codons (46%). The tRNA with the matching anticodon CAC is
overrepresented over the other two isoforms. A similar observation can be made for glycine, which is
coded by CAA and CAG but the thermodynamically more stable CAG is favored and the tRNA with a a
CTG anticodon is more abundantly expressed.

Slide 26
Codon usage may be different in different organisms and is not universal. Part of the explanation is
the constraints of co-evolution between codons and anticodon tRNAs but other factors may also play a
role (e.g. environmental temperature). As shown here for the six Arginine codons, the preferred use of
these is vastly different in humans, Drosophila, and E. coli. Thus, if you want to efficiently express a
protein from one species in another, eventually, you have to optimize the gene sequence for codon
usage in the target species.

Slide 27
Even though we have seen examples that certain regulatory properties are hardwired into the genetic
code, such as control of transcription and binding efficiency of the ribosome for different start sites,
translation, is also actively regulated.

Slide 28
In prokaryotes, the most critical site of regulation is the ribosome binding site. The binding site could
be blocked by a regulatory protein that makes the site unavailable / unrecognizable by the small
ribosomal subunit. Alternatively, secondary structure of the mRNA may also prevent ribosomes from
binding. Often these secondary structures can be broken if a ribosome already passed through them
once.

Slides 29/30
An interesting case is the regulation of ribosomal proteins of which there are 21 and 34 that are part of
the small and large ribosomal subunits, respectively. These proteins are translated from several
multicistronic mRNAs. For all these RNAs, one protein encoded by one of the later cistrons (indicated
in red) typically regulates translation.

Slide 31
The multiple cistrons are translated by translational coupling, rather than by independent ribososme
binding sites. A single ribosome binding site exists at the beginning of the mRNA and the sequence
AUGA between cistrons doubles as a STOP (UGA) and a START (AUG) site. In this example, L4
regulates protein expression from all of the 11 cistrons encoded by the S10 operon (named after the
first cistron in the array). If the amount of L4 protein is high inside the bacterial cells, it binds near the
RBS and prevents the recognition by the small ribosomal subunit. By blocking the initial binding event,
all of the later cistrons are also not translated because of the coupling. This is an example where the
absolute amount of protein regulates translation.

Slides 32/33
The regulation can be more sophisticated as in the case shown here. It is critical that the amount of
ribosomal proteins is balanced with the abundance of the respective ribosomal RNAs. For instance,
the ribosomal protein S8 binds both to the 16S rRNA and to the transcript of the spc operon that
includes S8. The S8 cistron forms a hairpin loop structure that includes the ribosome binding site.
When more rRNA than S8 protein is found in the cell, S8 will bind to the 16S rRNA. But after all
binding sites are occupied, it will also bind to a lower affinity site at the beginning of the mRNA and
prevent translation of the included proteins. Translation by the ribosome is needed here to free the
ribosome binding site of S8, which does not happen in this case.

Slide 34
In eukaryotes recognition of the 5’-end of the mRNA and assembly of the 43S initiation complex are
independent events. Both are critical steps during translation that can be regulated. Several
circumstances, such as nutrient starvation or ER stress can turn down translation globally because the
cell does not have the necessary resources available. These events result in the activation of eIF2a
kinases. Phosphorylation of eIF2 reduces its activity and, thus, formation of the 43S complex, which
results in reduced general translation.

Slides 35/36
Another important switch of translation is the AKT/mTOR pathway. Proteins that bind the initiation
factor 4E function (4E-BP) as inhibitors of mRNA recognition because the overlap with binding sites
for eIF4G. mTOR phosphorylates 4E-BPs, which allows translation to initiate since the phosphorylated
forms of the protein cannot bind to 4E. This pathway is often activated downstream of growth factor
receptors. The receptor activates AKT, which inhibits a protein that inhibits mTOR. Thus, when the
receptor is activated, mTOR is disinhibited and protein translation can result.

Slides 37/38
In the previous example 4E-BPs would interact with any mRNA, thus protein translation is regulated
globally. Yet, there are also cases in which translation of individual mRNAs is regulated by 4E-BPs.
We will see more details in later lectures but during Drosophila embryogenesis, a spatial pattern of
gene expression and protein translation is established that defines distinct anatomical positions of the
embryo. One such protein is the Oscar protein that is only expressed in the caudal end of the oocyte.
However, the mRNA is deposited into the oocyte by nurse cells at the opposite anterior pole. The
mRNA is prevented from being translated along its transport across the oocyte by specific 4E-BP that
are recruited to the mRNA (by Bruno).

End of transcript !!! - (shf, 03.12.2021)