Lecture 3: Translation and Mutations

Learning Goals:

Students should understand:

• How mRNA is translated to produce a polypeptide.

• The terms that geneticists use to refer to different types of mutations.
• How exogenous factors can cause DNA damage.
• How endogenous factors can cause mutation.
• How limitations of the DNA replication machinery lead to mistakes in
repetitive DNA sequences.

Students should be able to:

• Differentiate between the types of mutations that commonly

occur (e.g. deletions, insertions, & single base changes).
• Predict which mutations are permissible and which are not, on
the basis of the genetic code.
• Identify common external sources of mutations (ionizing
radiation, UV radiation & chemical agents, such as alkylating
agents).
• Identify biological sources of mutations (transposons &
replication slippage).
• Predict the type(s) of mutation(s) that a given mutagen could
cause (i.e., identify a mutagen based on the type of mutation
that is found in an individual that was exposed to it).
Lecture Notes:

Translation
We finished last lecture with a discussion of transcription and the
ways in which this process is regulated. In the next phase, translation, the
cells take the information transcribed from DNA into RNA and turns it into
active biological molecules, proteins.
After the discovery of the DNA double helix, attention turned to how
the four bases of DNA could encode the 20 different amino acids that
make up proteins. A key experiment was done by Francis Crick and
Sydney Brenner. By inducing insertion or deletion mutations in the DNA
sequence, they found that mutations were much less disruptive if they
added or subtracted three base pairs, rather than just one or two. They
concluded that the code of DNA is read in triplets, three base pairs at a
time. Adding or removing one or two base pairs caused a frameshift
mutation, whereby all downstream information was out of phase
(meaning that the wrong amino acids would be incorporated into the
protein). However, adding or removing three base pairs simply added or
removed a single amino acid, with the rest of the sequence remaining in
phase.
Now that we knew that base pair triplets corresponded to amino
acids, the next step was to find which amino acid each of the 64 possible
triplets (codons) correspond to. A key step came when Marshall Nirenberg
synthesized a string of RNA U’s (i.e. UUUUUUUU…) and added this to a
cellular extract capable of producing protein. The result was a simple
polypeptide consisting of a string of the amino acid phenylalanine: UUU
encodes Phe (to use the three letter amino acid abbreviation for
phenylalanine). Over the next several years, the rest of the genetic code
was determined. Further study showed the code is, more or less, a
universal feature of all living systems.

Molecular mechanism of translation

In eukaryotes, mRNA is exported from the nucleus (via nuclear
pores). In the cytoplasm, translation takes place in the ribosome, an
RNA/protein complex composed of a large and a small subunit. The
other key players are transfer RNAs (tRNA), each between 70 and 90
nucleotides long, with a characteristic self-pairing structure that can be
represented schematically as a cloverleaf. At one end of tRNA is the anti-
codon, the three nucleotides that undergo pairing with the corresponding
codon in the mRNA. At the other end is the point of attachment for an
amino acid, the one that corresponds to the molecule’s anti-codon. An
enzyme called aminoacyl tRNA synthetase connects specific amino acids
to their appropriate tRNA molecule, via a covalent bond. Most organisms
have one aminoacyl tRNA synthetase for each amino acid. A tRNA not
bound to an amino acid is an uncharged tRNA and one that is bound to
an amino acid is a charged tRNA.
There are three separate phases of translation: initiation, elongation
and termination. During translation initiation in eukaryotes, initiation
factors, a special “initiator” tRNA charged with methionine, and the small
subunit of a ribosome all assemble at the 5’ cap of the mRNA. This
complex, which is called the initiation complex, scans the mRNA for an
AUG start codon, moving along the mRNA from the 5’ towards the 3’ end.
When this complex encounters an AUG, the initiator tRNA, which has the
anticodon 5’-CAU-3’, can base pair with it. This sets the start site for
translation. Note that even though AUG is the first codon to be translated,
it is not typically the first codon in the mRNA: there is a preceding region of
mRNA that is not translated. Once the initiation complex has established
the connection to the AUG codon, the large subunit of the ribosome joins.
The polypeptide is synthesized from the amino end to the carboxyl end,
meaning that methionine will be the amino acid at the amino end of the
polypeptide. Note that the AUG codon is critical for the establishment of
the proper reading frame.
The next phase is elongation. The large subunit contains three tRNA-
accommodating positions. The initial methionine tRNA is located in the
central peptidyl site (P-site). The next tRNA (i.e. the one whose anti-codon
corresponds to the contiguous codon on the mRNA) arrives in the
aminoacyl site (A-site). With the two tRNA’s sitting beside each other, the
peptide bond between the two amino acids is catalyzed, with the
methionine detaching from its tRNA, and joining to the new amino acid,
which, for now, remains attached to its tRNA. Then, the ribosome shifts
along one codon, moving the uncharged methionine tRNA into the exit
site (E-site) of the ribosome and the second tRNA to the peptidyl position.
The tRNA in the E-site is ejected, and the two amino acid dipeptide
attached to the tRNA in the P-site forms a peptide bond with the newly
arrived amino acid in the A-site, creating a three amino acid polypeptide.
Everything shifts along one codon. The process then repeats to generate
a growing chain of amino acids, as specified by the mRNA.
The final phase, termination, occurs when the process encounters a
stop codon for which there are no corresponding tRNA’s. Instead, a
release factor binds to the aminoacyl site of the ribosome. This causes the
bond connecting the polypeptide to the tRNA in the peptidyl site to
break, releasing the protein.

Because of the redundancy of the genetic code (some amino acids are
encoded by as many as six codons), we can categorize mutations in
coding DNA as either synonymous [or silent] (they change the DNA
sequence, but not the resulting amino acid sequence) or nonsynonymous
[or replacement] (they change the DNA sequence and the resulting
amino acid sequence).

A glimpse of the future? Leveraging the redundancy of the genetic code

to generate codons specifying alternative amino acids
Living systems use 20 amino acids. Biology may be limited to 20
amino acids, but chemistry most certainly is not. Many other amino acids
exist, and still others can be synthesized by organic chemists. Some of
these “alternative” (or “non-canonical” because the standard 20 amino
acids are deemed “canonical”) amino acids have unusual properties
which may be useful when the amino acid is incorporated into a protein.
But how can we create a living system capable to using such non-
canonical amino acids?
Since all the 64 possible codons have a specific meaning (i.e., code
for a canonical amino acid or are stop codons), the main problem that
we have to solve is removing the meaning of some of the codons, so we
can then use them to encode non-canonical amino acids. Here, the
redundancy of the genetic code helps us. For example, six codons code
for serine, a canonical amino acid. A recent study reduced this number in
E. coli to four by replacing two of the serine codons throughout the entire
genome of E. coli with other serine codons. The serine codons that
researchers removed were TCG and TCA. They changed every TCG in
the E. coli genome to AGC, another serine codon, and changed every
TCA to AGT, again another serine codon. The mechanics of this editing
process were surprisingly simple because we now have effective
chemical technologies that allow us to synthesize DNA molecules. The
researchers simply synthesized an entire E. coli genome with the codon
substitutions in place – that was a total of 18,214 edited codons! Note
that this search-and-replace process throughout the E. coli genome did
not change a single amino acid: every serine encoded in the original
genome is still a serine in the recoded genome. But now TCG and TCA
are eliminated from the E. coli genome, and replaced by AGC and AGT.
This means that we can now dedicate each of these codons, TCG and
TCA, to a new amino acid, even a non-canonical one. Needless to say,
there is still a long way to go with this technology: we need in addition to
create transfer RNAs corresponding to our TCG or TCA codons that are
coupled to the non-canonical amino acid(s) we aim to incorporate into
the protein. A long way to go, for sure, but a remarkable first step,
regardless.

Mutation
Researchers perform genetic analyses in many ways, but a theme
that is common to all scientists who use a genetic approach to answer
biological questions is the study of genetic variants – individuals with
different versions of genes that are important for the process under
investigation. In many cases, these experiments rely on the identification
of mutations that affect gene functions and, consequently, any biological
processes that involve the mutated gene.
For example, classic studies in fields such as developmental
genetics involve the identification of mutants with specific phenotypes of
interest. The essential strategy is to “break” a gene by making a mutation
in it, then see what effect this has on the process of interest (see
http://www2.biology.ualberta.ca/locke.hp/dougandbill.htm for a story
that summarizes the geneticist’s approach to answering biological
questions). Therefore, if an individual is interested in learning how insect
wings develop, they will look for genes that, when mutated, cause wings
to be abnormal. Researchers then perform further analyses to determine
why loss of these genes’ functions have the effects that they do.
Mutations are not only important for research, though – the accumulation
of mutations over time is how diversity arises, and is necessary for evolution
to occur.
We’re going to be looking at several different questions that all
relate to mutations:
• Where do they come from?
• What effect do they have on genes?
• How can we detect them?
• How can we characterize them?

In some cases, the mutants that researchers study are found by

happenstance, but often researchers deliberately introduce mutations
using DNA damaging agents in order to obtain a sufficient number and
variety of mutants to study. The process of inducing mutations – either
randomly or in a directed way – is known as mutation. This can be
defined as the introduction of a change in a DNA sequence in a way that
means it will be passed on to the next generation of cells or organisms.
The specific changes that occur are known as mutations. The term
“mutation” refers both to the process by which these changes occur
(usually damage to the DNA that goes uncorrected), as well as the
products of this process.

Somatic versus germline mutations

Mutations can occur in any cell in the body. However, depending
on which cell types they occur in, the effect of these mutations can vary.
When they occur in the somatic cells, they are limited in their effect to
those cells and their direct descendants – in other words, they are not
passed on to the offspring, and they only affect a subset of cells in the
body of the individual in which they occurred. This is not to say that
somatic mutations are unimportant: they may, for example, cause
cancer. When mutations occur in germ cells, they can be passed on to
the next generation, with the offspring’s every cell being affected.

Types of mutations and mutagens

Depending on the type of DNA damage, mutations occur on
different scales. Many mutations involve changes to a single base in the
DNA sequence or a small number of bases, and so affect only the specific
gene whose sequence is altered. These are often referred to as gene
mutations, because they affect a single gene. Other mutations involve
larger-scale changes in the structure or number of chromosomes. For
example, large insertions or deletions that affect many genes and
chromosomal translocations (in which a piece of one chromosome is
fused to a different chromosome) are chromosomal mutations (or
chromosomal rearrangements).
Mutations can also be classified depending on their origin. Those
that are caused by exposure to a specific DNA damaging agent, or
mutagen (such as UV light, chemical agents, biological agents, etc.) are
said to be induced. Those that occur during normal biological processes
such as DNA replication or cell division are considered spontaneous.
Different types of DNA damaging agents tend to cause different
types of mutations. Many chemicals will cause smaller-scale gene
mutations, while certain types of radiation are more likely to induce large-
scale chromosomal mutations.

Induced Mutations
UV Radiation
One common mutagen is UV radiation from exposure to sunlight
and artificial sources. UV radiation is limited in its effect in two ways. First,
it cannot pass through our skin, so that it can only cause mutations in cells
that are directly exposed to the sun. This is why sun exposure is known to
increase an individual’s risk of developing skin cancer, but not stomach
cancer. In addition, UV radiation can cause a very specific type of DNA
damage, which is a covalent bond between two adjacent pyrimidine
bases (such as TT, for example). If left unrepaired, this DNA damage can
cause errors during DNA replication, leading to changes at the level of
one or two base pairs.

Ionizing Radiation
In contrast to UV radiation, ionizing radiation can penetrate deeply
into tissues, and so causes mutations throughout the body, instead of only
in the skin. It can cause DNA damage in a variety of ways, including
damaging bases and causing breaks in the sugar-phosphate backbone.
Damaged bases can mispair, causing small-scale gene mutations, while
double-stranded breaks can fragment chromosomes, causing
chromosomal mutations. When the chromosome fragments are put back
together, a number of different chromosomal rearrangements can occur.
These include small- and large-scale deletions and duplications,
inversions, insertions, and translocations that can affect hundreds or even
thousands of genes.

Chemical Agents
Like those caused by ionizing radiation, mutations caused by
chemical agents can affect all cells in the body. This is because chemical
agents can circulate in the blood and lymph, so that all cells, including
both somatic and germ cells, are exposed. However, unlike ionizing
radiation, chemical agents are generally limited to causing smaller-scale
gene mutations, because they affect individual bases.
One type of chemical mutagen is alkylating agents. These
chemicals add bulky hydrocarbon groups to DNA bases, affecting their
ability to base-pair. One type of alkylating agent is mustard gas, which
was used during World War I. Exposure to this mutagen causes such
severe DNA damage that cells of affected individuals die, causing
blistering, bleeding, and other tissue damage. Survivors are also at an
increased risk of developing cancer. Alkylating agents are used by
scientists to induce mutations in experimental organisms (because, as
noted above, it’s possible to derive information about the function of a
gene by disrupting that function mutationally). For example,
ethylmethylsulfonate (EMS) changes guanine to produce a modified
nucleotide which pairs with thymine. Thus, EMS produces C • G → T • A
changes.

Spontaneous mutations
So far, we have focused our discussion on different environmental
factors that can induce mutations. While these mutagens are important to
understand and can contribute significantly to the introduction of
mutations in an individual’s DNA, there are also many mutations that
occur spontaneously.

Depurination
Mutations may result from spontaneous chemical changes in DNA.
One such change is depurination, the loss of a purine base [i.e., an A or a
G] from a nucleotide. Depurination results when the covalent bond
connecting the purine to the deoxyribose sugar breaks, producing an
apurinic site, a nucleotide that lacks its purine base. An apurinic site
cannot act as a template for a complementary base in replication. In the
absence of base-pairing constraints, an incorrect nucleotide (usually
adenine) is incorporated into the newly synthesized DNA strand opposite
the apurinic site. Pyrimidines are less prone to this kind of spontaneous
decay.

Transposons
Biological agents such as transposons (transposable elements) can
cause mutations. Transposons are mobile genetic elements that can
either copy and paste themselves into new sites in the genome or cut
themselves out of their current location and insert into a new one. They
were first discovered by Barbara McClintock in her studies of the
pigmentation of corn kernels. When transposons insert themselves, they
can disrupt the coding or regulatory sequences of genes, which can
interfere with their functions. An example of the mutagenic effect of
transposable elements is seen in the color of grapes. Black and red grapes
result from the production of red pigments, called anthocyanins, which
are lacking in white grapes. White grapes resulted from a mutation
caused by the insertion of a transposable element into the anthocyanin-
producing gene in black grapes that turned off the production of
anthocyanins. Red grapes resulted from a second mutation that occurred
in white grapes removing most, but not all, of the transposon. This
switched anthocyanin production back on, but not as intensely as in the
original black grapes. In other cases, transposons insert into noncoding
regions and do not cause any phenotypic consequences. Genomes
bear testimony to past bouts of transposon activity: around 10% of the
human genome consists of copies of a particular element, Alu, which is no
longer actively undergoing transposition, but which was clearly highly
active in the past.
The life cycles of transposons vary. Many are retrotransposons that
go through an RNA phase: RNA is transcribed and then converted into
DNA by the enzyme reverse transcriptase before the DNA segment is
incorporated into a new site in the genome.

Errors in DNA Replication

An important source of mutation is when DNA polymerase makes a
mistake and it goes uncorrected.
Overall error rates have been estimated for many different DNA
polymerases. While the exact number varies for different DNA
polymerases, the rates are often approximately 1 error (i.e., incorporation
of a mis-matched base) for every 106 bases. Cells can often recognize
these mis-incorporated bases because they distort the DNA double helix
due to their incorrect base-pairing and fix them. A reasonable estimate
for the overall error rate of DNA synthesis is 1 mis-incorporated base for
every 109 bases.
 In addition to these randomly mis-incorporated bases, certain types
of DNA sequence are prone to mutation. Specifically, short tandem
repeats – simple repetitive sequences such as ATATATAT or CAGCAGCAG
– accumulate changes at an accelerated rate. During DNA replication,
there is a tendency for these repetitive sequences to expand, or increase
in the number of copies of the repeats. These repeat expansions are
associated with many neurological diseases including amyotrophic lateral
sclerosis, Lou Gehrig’s Disease, and Huntington’s disease.
Concept Check:

Translation
Mutation
Somatic versus germline mutation
Chromosomal rearrangement
UV radiation
Ionizing radiation
Alkylating agent
Replication slippage
Transposons
Retrotransposons
Reverse transcriptase
Single/double strand breaks
Base change/substitution
Deletion
Duplication
Inversion
Insertion
Translocation
Depurination
Short tandem repeats
Repeat expansion
Spontaneous versus induced mutation