Genetic Codes
Genetic codes
A series of codons
in part of a mRNA
molecule. Each codon consists of three nucleotides,
representing a single amino acid.
The genetic code is the set of rules by
which information encoded in genetic material (DNA or RNA sequences) is translated into proteins
(amino acid
sequences) by living cells. Specifically, the code defines a mapping
between tri-nucleotide
sequences called codons and amino acids; every triplet of nucleotides in
a nucleic acid sequence specifies a single amino acid. Because the vast
majority of genes
are encoded with exactly the same code (see #RNA codon
table), this particular code is often referred to as the canonical
or standard genetic code, or simply the genetic code, though in fact
there are many variant codes; thus, the canonical genetic code is not
universal. For example, in humans, protein synthesis in mitochondria
relies on a genetic code that varies from the canonical code.
Cracking
the genetic code
After the structure of DNA was deciphered by James Watson,
Francis Crick,
Maurice
Wilkins and Rosalind Franklin, serious efforts to
understand the nature of the encoding of proteins began. George Gamov
postulated that a three-letter code must be employed to encode the 20 different
amino acids
used by living cells to encode proteins (because 3 is the smallest n such that
4n is at least 20). The fact that codons did consist of three DNA
bases was first demonstrated in the Crick, Brenner et al. experiment.
The first elucidation of a codon was done by Marshall Nirenberg and Heinrich J. Matthaei in 1961 at the National Institutes of Health. They used a
cell-free
system to translate a poly-uracil RNA sequence (or
UUUUU... in biochemical terms) and discovered that the polypeptide
they had synthesized consisted of only the amino acid phenylalanine.
They thereby deduced from this poly-phenylalanine that the codon UUU specified
the amino-acid phenylalanine. Extending this work, Nirenberg and his coworkers
were able to determine the nucleotide makeup of each codon. In order to
determine the order of the sequence, trinucleotides were bound to ribosomes and
radioactively labeled aminoacyl-tRNA was used to determine which amino acid
corresponded to the codon. Nirenberg's group was able to determine the
sequences of 54 out of 64 codons. Subsequent work by Har Gobind Khorana identified the rest of the
code, and shortly thereafter Robert W.
Holley determined the structure of transfer RNA,
the adapter molecule that facilitates translation. In 1968, Khorana, Holley and
Nirenberg shared the Nobel Prize in Physiology or Medicine for their
work.
Transfer of
information via the genetic code
The genome of an organism is inscribed in DNA, or in some viruses RNA. The portion of the
genome that codes for a protein or an RNA is referred to as a gene. Those genes that
code for proteins are composed of tri-nucleotide units called codons,
each coding for a single amino acid. Each nucleotide sub-unit consists of a phosphate,
deoxyribose
sugar and one of the 4 nitrogenous nucleotide
bases. The purine
bases adenine
(A) and guanine
(G) are larger and consist of two aromatic rings. The pyrimidine
bases cytosine
(C) and thymine
(T) are smaller and consist of only one aromatic ring. In the
double-helix configuration, two strands of DNA are joined to each other by
hydrogen bonds in an arrangement known as base pairing.
These bonds almost always form between an adenine base on one strand and a
thymine on the other strand and between a cytosine base on one strand and a
guanine base on the other. This means that the number of A and T residues will
be the same in a given double helix as will the number of G and C residues. In
RNA, thymine (T) is replaced by uracil (U),
and the deoxyribose is substituted by ribose.
Each protein-coding gene is transcribed into a template molecule of
the related polymer RNA, known as messenger RNA
or mRNA. This in turn is translated on the ribosome
into an amino acid
chain or polypeptide.
The process of translation requires transfer RNAs
specific for individual amino acids with the amino acids covalently
attached to them, guanosine triphosphate as an energy
source, and a number of translation factors. tRNAs have anticodons
complementary to the codons in mRNA and can be "charged" covalently
with amino acids at their 3' terminal CCA ends. Individual tRNAs are charged
with specific amino acids by enzymes known as aminoacyl tRNA synthetases which have high
specificity for both their cognate amino acids and tRNAs. The high specificity
of these enzymes is a major reason why the fidelity of protein translation is
maintained.
There are 4³ = 64 different codon combinations
possible with a triplet codon of three nucleotides. In reality, all 64 codons
of the standard genetic code are assigned for either amino acids or stop
signals during translation. If, for example, an RNA sequence, UUUAAACCC is
considered and the reading-frame starts with the first U (by
convention, 5' to 3'), there are three codons, namely, UUU, AAA and CCC,
each of which specifies one amino acid. This RNA sequence will be translated
into an amino acid sequence, three amino acids long.
The standard genetic code is shown in the
following tables. Table 1 shows what amino acid each of the 64 codons
specifies. Table 2 shows what codons specify each of the 20 standard amino
acids involved in translation. These are called forward and reverse codon
tables, respectively. For example, the codon AAU represents the amino acid asparagine,
and UGU and UGC represent cysteine (standard three-letter designations, Asn and Cys
respectively).
Reading
frame of a sequence
Note that a codon is defined by the
initial nucleotide from which translation starts. For example, the string
GGGAAACCC, if read from the first position, contains the codons GGG, AAA and
CCC; and if read from the second position, it contains the codons GGA and AAC;
if read starting from the third position, GAA and ACC. Partial codons have been
ignored in this example. Every sequence can thus be read in three reading
frames, each of which will produce a different amino acid
sequence (in the given example, Gly-Lys-Pro, Gly-Asp, or Glu-Thr,
respectively). With double-stranded DNA there are six possible reading
frames, three in the forward orientation on one strand and three
reverse (on the opposite strand).
The actual frame a protein sequence is
translated in is defined by a start codon,
usually the first AUG codon in the mRNA sequence. Mutations that disrupt the
reading frame by insertions or deletions of a non-multiple of 3 nucleotide
bases are known as frameshift mutations. These mutations may
impair the function of the resulting protein, if it is formed, and are thus
rare in in vivo
protein-coding sequences. Often such misformed proteins are targeted for
proteolytic degradation. In addition, a frame shift mutation is very likely to
cause a stop codon to be read which truncates the creation of the protein
(example). One reason for the rareness of frame-shifted mutations being
inherited is that if the protein being translated is essential for growth under
the selective pressures the organism faces, absence of a functional protein may
cause lethality before the organism is viable.
Translation starts with a chain initiation
codon (start codon). Unlike stop codons, the codon alone is not
sufficient to begin the process. Nearby sequences and initiation
factors are also required to start translation. The most common
start codon is AUG, which codes for methionine, so most amino acid chains start
with methionine.
The three stop codons
have been given names: UAG is amber, UGA is opal (sometimes also
called umber), and UAA is ochre. "Amber" was named by
discoverers Richard Epstein and Charles Steinberg after their friend Harris
Bernstein, whose last name means "amber" in German. The other two
stop codons were named 'ochre" and "opal" in order to keep the
"color names" theme. Stop codons are also called termination codons
and they signal release of the nascent polypeptide from the ribosome due to
binding of release factors in the absence of cognate tRNAs
with anticodons complementary to these stop signals.
Degeneracy of the genetic
code
The genetic code has redundancy but no
ambiguity. For example, although codons GAA and GAG both specify glutamic acid
(redundancy), neither of them specifies any other amino acid (no ambiguity).
Degenerate codons may differ in their third positions; e.g., both GAA and GAG
code for the amino acid glutamic acid. A codon is said to be fourfold
degenerate if any nucleotide at its third position specifies the same amino
acid; it is said to be twofold degenerate if only two of four possible
nucleotides at its third position specify the same amino acid. In twofold
degenerate codons, the equivalent third position nucleotides are always either
two purines
(A/G) or two pyrimidines (C/T). Only two amino acids are specified by a
single codon; one of these is the amino-acid methionine,
specified by the codon AUG, which also specifies the start of translation; the
other is tryptophan,
specified by the codon UGG. The degeneracy of the genetic code is what accounts
for the existence of silent mutations.
Degeneracy results because a triplet code
designates 20 amino acids and a stop codon. Because there are four bases,
triplet codons are required to produce at least 21 different codes. For
example, if there were two bases per codon, then only 16 amino acids could be
coded for (4²=16). Because at least 21 codes are required, then 4³ gives 64
possible codons, meaning that some degeneracy must exist.
These properties of the genetic code make it
more fault-tolerant for point mutations. For example, in theory,
fourfold degenerate codons can tolerate any point mutation at the third
position, although codon usage bias restricts this in practice in
many organisms; twofold degenerate codons can tolerate one out of the three
possible point mutations at the third position. Since transition mutations (purine to purine or
pyrimidine to pyrimidine mutations) are more likely than transversion
(purine to pyrimidine or vice-versa) mutations, the equivalence of purines or
that of pyrimidines at twofold degenerate sites adds a further fault-tolerance.
A practical consequence of redundancy is that
some errors in the genetic code only cause a silent mutation or an error that
would not affect the protein because the hydrophilicity
or hydrophobicity
is maintained by equivalent substitution of amino acids; for example, a codon
of NUN (where N = any nucleotide) tends to code for hydrophobic amino acids.
Even so, it is a single point mutation that causes a modified hemoglobin
molecule in sickle-cell disease. The hydrophilic glutamate
(Glu) is substituted by the hydrophobic valine (Val), which
reduces the solubility of ß-globin. In this case, this mutation causes hemoglobin
to form linear polymers linked by the hydrophobic interaction between the
valine groups causing sickle-cell deformation of erythrocytes. Sickle-cell
disease is generally not caused by a de novo mutation.
Rather it is selected for in malarial regions (in a way similar to thalassemia),
as heterozygous
people have some resistance to the malarial Plasmodium
parasite (heterozygote advantage).
These variable codes for amino acids are allowed
because of modified bases in the first base of the anticodon
of the tRNA, and the base-pair formed is called a wobble base
pair. The modified bases include inosine
and the Non-Watson-Crick U-G basepair.
Variations to the
standard genetic code
While slight variations on the standard code had
been predicted earlier, none were discovered until 1979, when researchers
studying human mitochondrial genes discovered they
used an alternative code. Many slight variants have been discovered since,
including various alternative mitochondrial codes, as well as small variants
such as Mycoplasma
translating the codon UGA as tryptophan. In bacteria
and archaea,
GUG and UUG are common start codons. However, in rare cases, certain specific
proteins may use alternative initiation (start) codons not normally used by
that species.
In certain proteins, non-standard amino acids
are substituted for standard stop codons, depending upon associated signal
sequences in the messenger RNA: UGA can code for selenocysteine
and UAG can code for pyrrolysine as discussed in the relevant
articles. Selenocysteine is now viewed as the 21st amino acid, and pyrrolysine
is viewed as the 22nd. A detailed description of variations in the genetic code
can be found at the NCBI web site.
However, all known codes have strong
similarities to each other, and the coding mechanism is the same for all
organisms: three-base codons, tRNA, and ribosomes, reading the code in the same
direction, translating the code three letters at a time into sequences of amino
acids.
Theories
on the origin of the genetic code
Despite the variations that exist, the genetic
codes used by all known forms of life on Earth are very similar. Since there
are many possible genetic codes that are thought to have similar utility to the
one used by Earth life, the theory of evolution
suggests that the genetic code was established very early in the history of
life and meta-analysis of transfer RNA suggest it was established soon after
the formation of earth.
One can ask the question: is the genetic code
completely random, just one set of codon-amino acid correspondences that
happened to establish itself and be "frozen in" early in evolution,
although functionally any of the many other possible transcription
tables would have done just as well? Already a cursory look at the table shows
patterns that suggest that this is not the case.
There are three themes running through the many
theories that seek to explain the evolution of the genetic code (and hence the
origin of these patterns). One is illustrated by recent aptamer
experiments which show that some amino acids have a selective chemical affinity
for the base triplets that code for them. This suggests that the current,
complex translation mechanism involving tRNA and associated
enzymes may be a later development, and that originally, protein sequences were
directly templated on base sequences. Another is that the standard genetic code
that we see today grew from a simpler, earlier code through a process of
"biosynthetic expansion". Here the idea is that primordial life
'discovered' new amino acids (e.g. as by-products of metabolism) and later
back-incorporated some of these into the machinery of genetic coding. Although
much circumstantial evidence has been found to suggest that fewer different
amino acids were used in the past than today, precise and detailed hypotheses
about exactly which amino acids entered the code in exactly what order has
proved far more controversial. A third theory is that natural
selection has led to codon assignments of the genetic code that
minimize the effects of mutations.
PCR optimization
The polymerase chain reaction (PCR) is a
commonly used molecular biology tool for amplifying DNA, and various techniques
for PCR optimization have been developed by molecular biologists to
improve PCR performance and minimize failure.
Contamination
and PCR
The PCR method is extremely sensitive, requiring
only a few DNA molecules in a single reaction for amplification across several
orders of magnitude. Therefore, adequate measures to avoid contamination from
any DNA present in the lab environment (bacteria,
viruses,
or human sources) are required. Because products from previous PCR
amplifications are a common source of contamination, many molecular biology
labs have implemented procedures that involve dividing the lab into separate
areas. One lab area is dedicated to preparation and handling of pre-PCR
reagents and the setup of the PCR reaction, and another area to post-PCR
processing, such as gel electrophoresis or PCR product
purification. For the setup of PCR reactions, many standard operating procedures involve
using pipettes
with filter tips and wearing fresh laboratory
gloves, and in some cases a laminar flow cabinet with UV lamp as a
work station (to destroy any extraneous DNA before PCR setup). Possible
contamination with extraneous DNA or primer-multimer formation is routinely
assessed with a (negative) control PCR reaction. This control reaction is set
up in the same way as the experimental PCRs, but without template DNA added,
and is performed alongside the experimental PCRs.
Secondary structures in the DNA can result in
folding or knotting of DNA template or primers, leading to decreased product
yield or failure of the reaction. Hairpins, which consist of internal folds
caused by base-pairing between nucleotides in inverted repeats within
single-stranded DNA, are common secondary structures and may result in failed
PCRs.
Typically, primer design that includes a check
for potential secondary structures in the primers, or addition of DMSO or glycerol
to the PCR to minimize secondary structures in the DNA template [citation needed], are used in
the optimization of PCRs that have a history of failure due to suspected DNA
hairpins.
Taq
polymerase lacks a 3' to 5' exonuclease activity. Thus, Taq has no
error-proofreading activity, which consists of excision of any newly
polymerized nucleotide base from the nascent (=extending) DNA strand that does
not match with its opposite base in the complementary DNA strand. The lack in
3' to 5' proofreading of the Taq enzyme results in a high error rate
(approximately 1 in 10,000 bases), which affects the fidelity of the PCR, especially
if errors occur early in the PCR, causing accumulation of a large proportion of
amplified DNA with incorrect sequence in the final product.
Several "high-fidelity" DNA
polymerases, having engineered 3' to 5' exonuclease activity, have become available
that permit more accurate amplification for use in PCRs for sequencing or
cloning of products. Examples of polymerases with 3' to 5' exonuclease activity
include: KOD DNA polymerase, a recombinant form of Thermococcus
kodakaraensis KOD1; Vent, which is extracted from Thermococcus litoralis;
Pfu DNA polymerase, which is extracted from Pyrococcus furiosus; and Pwo, which is
extracted from Pyrococcus woesii.[citation needed]
PCR works readily with DNA of up to two to three
thousand base pairs in length. However, above this size, product yields often
decrease, as with increasing length stochastic
effects such as premature termination by the polymerase begin to affect the
efficiency of the PCR. It is possible to amplify larger pieces of up to 50,000
base pairs with a slower heating cycle and special polymerases. These are
polymerases fused to a processivity-enhancing DNA-binding
protein, enhancing adherence of the polymerase to the DNA.
Other valuable properties of the chimeric
polymerases TopoTaq and PfuC2 include
enhanced thermostability, specificity and resistance to contaminants and
inhibitors[3][4].
They were engineered using unique Helix-hairpin-Helix (HhH) DNA binding domains
of Topoisomerase
V from hyperthermophile Methanopyrus kandleri. Chimeric polymerases
overcome many limitations of native enzymes and are used in direct PCR
amplification from cell cultures and even food samples, thus by-passing
laborious DNA isolation steps. A robust strand-displacement activity of the
hybrid TopoTaq polymerase helps solving PCR problems with hairpins
and G-loaded double helices, because helices with a
high G-C context possess a higher melting temperature.
Non-specific binding of primers frequently
occurs and can be due to repeat sequences in the DNA template, non-specific
binding between primer and template, and incomplete primer binding, leaving the
5' end of the primer unattached to the template. Non-specific binding is also
often increased when degenerate primers are used in the PCR.
Manipulation of annealing temperature and magnesium
ion (which stabilise DNA
and RNA
interactions) concentrations can increase specificity. Non-specific priming
during reaction preparation at lower temperatures can be prevented by using
"hot-start" polymerase enzymes whose active site is blocked by an
antibody or chemical that only dislodges once the reaction is heated to 95˚C
during the denaturation step of the first cycle.
A new way to maintain thermophilic enzymes
absolutely inactive at low temperature was identified during structural studies
of hyperthermophilic DNA-binding enzymes. A specially engineered TopoTaq polymerase
activates instantly at high temperature and overcomes limitations of
conventional "hot-start" enzymes that require antibody denaturation
at >90˚C for activation. In addition, its activity is blocked upon
completion of PCR at low temperature.
Other methods to increase specificity include Nested PCR
and Touchdown PCR.