DNA TRANSLATION
DNA to mRNA to Protein:
The genes in DNA encode protein molecules, which are the "workhorses" of
the cell, carrying out all the functions necessary for life. For example, enzymes,
including those that metabolize nutrients and synthesize new cellular
constituents, as well as DNA polymerases and other enzymes that make copies
of DNA during cell division, are all proteins.
In the simplest sense, expressing a gene means manufacturing its corresponding
protein, and this multilayered process has two major steps. In the first step, the
information in DNA is transferred to a messenger RNA (mRNA) molecule by
way of a process called transcription. During transcription, the DNA of a gene
serves as a template for complementary base-pairing, and
an enzyme called RNA polymerase II catalyses the formation of a pre-mRNA
molecule, which is then processed to form mature mRNA (Figure 1). The
resulting mRNA is a single-stranded copy of the gene, which next must be
translated into a protein molecule.
During transcription, the enzyme RNA polymerase (green) uses DNA as a
template to produce a pre-mRNA transcript (pink). The pre-mRNA is processed
to form a mature mRNA molecule that can be translated to build the protein
molecule (polypeptide) encoded by the original gene.
Figure 1: A gene is expressed through the processes of transcription and
translation.
Figure Detail
During translation, which is the second major step in gene expression, the
mRNA is "read" according to the genetic code, which relates the DNA sequence
to the amino acid sequence in proteins (Figure 2). Each group of three bases in
mRNA constitutes a codon, and each codon specifies a particular amino acid
(hence, it is a triplet code). The mRNA sequence is thus used as a template to
assemble—in order—the chain of amino acids that form a protein.
Figure 2: The amino acids specified by each mRNA codon. Multiple codons
can code for the same amino acid.
The codons are written 5' to 3', as they appear in the mRNA. AUG is an
initiation codon; UAA, UAG, and UGA are termination (stop) codons.
Figure Detail
But where does translation take place within a cell? What individual substeps
are a part of this process? And does translation differ between prokaryotes and
eukaryotes? The answers to questions such as these reveal a great deal about the
essential similarities between all species.
Where Translation Occurs
Within all cells, the translation machinery resides within a specialized organelle
called the ribosome. In eukaryotes, mature mRNA molecules must leave
the nucleus and travel to the cytoplasm, where the ribosomes are located. On the
other hand, in prokaryotic organisms, ribosomes can attach to mRNA while it is
still being transcribed. In this situation, translation begins at the 5' end of the
mRNA while the 3' end is still attached to DNA.
In all types of cells, the ribosome is composed of two subunits: the large (50S)
subunit and the small (30S) subunit (S, for svedberg unit, is a measure of
sedimentation velocity and, therefore, mass). Each subunit exists separately in
the cytoplasm, but the two join together on the mRNA molecule. The ribosomal
subunits contain proteins and specialized RNA molecules—
specifically, ribosomal RNA (rRNA) and transfer RNA (tRNA).
Within the ribosome, the mRNA and aminoacyl-tRNA complexes are held
together closely, which facilitates base-pairing. The rRNA catalyzes the
attachment of each new amino acid to the growing chain.
The Beginning of mRNA Is Not Translated
Interestingly, not all regions of an mRNA molecule correspond to particular
amino acids. In particular, there is an area near the 5' end of the molecule that is
known as the untranslated region (UTR) or leader sequence. This portion of
mRNA is located between the first nucleotide that is transcribed and the start
codon (AUG) of the coding region, and it does not affect the sequence of amino
acids in a protein (Figure 3).
So, what is the purpose of the UTR? It turns out that the leader sequence is
important because it contains a ribosome-binding site. In bacteria, this site is
known as the Shine-Dalgarno box (AGGAGG), after scientists John Shine and
Lynn Dalgarno, who first characterized it. A similar site in vertebrates was
characterized by Marilyn Kozak and is thus known as the Kozak box. In
bacterial mRNA, the 5' UTR is normally short; in human mRNA, the median
length of the 5' UTR is about 170 nucleotides. If the leader is long, it may
contain regulatory sequences, including binding sites for proteins, that can
affect the stability of the mRNA or the efficiency of its translation.
Figure 3: A DNA transcription unit.
A DNA transcription unit is composed, from its 3' to 5' end, of an RNA-coding
region (pink rectangle) flanked by a promoter region (green rectangle) and a
terminator region (black rectangle). Regions to the left, or moving towards the
3' end, of the transcription start site are considered \"upstream;\" regions to the
right, or moving towards the 5' end, of the transcription start site are considered
\"downstream.\"
Translation Begins After the Assembly of a Complex Structure
The translation of mRNA begins with the formation of a complex on the mRNA
(Figure 4). First, three initiation factor proteins (known as IF1, IF2, and IF3)
bind to the small subunit of the ribosome. This preinitiation complex and a
methionine-carrying tRNA then bind to the mRNA, near the AUG start codon,
forming the initiation complex.
Figure 4: The translation initiation complex.
When translation begins, the small subunit of the ribosome and an initiator
tRNA molecule assemble on the mRNA transcript. The small subunit of the
ribosome has three binding sites: an amino acid site (A), a polypeptide site (P),
and an exit site (E). The initiator tRNA molecule carrying the amino acid
methionine binds to the AUG start codon of the mRNA transcript at the
ribosome’s P site where it will become the first amino acid incorporated into the
growing polypeptide chain. Here, the initiator tRNA molecule is shown binding
after the small ribosomal subunit has assembled on the mRNA; the order in
which this occurs is unique to prokaryotic cells. In eukaryotes, the free initiator
tRNA first binds the small ribosomal subunit to form a complex. The complex
then binds the mRNA transcript, so that the tRNA and the small ribosomal
subunit bind the mRNA simultaneously.
Figure Detail
Although methionine (Met) is the first amino acid incorporated into any new
protein, it is not always the first amino acid in mature proteins—in many
proteins, methionine is removed after translation. In fact, if a large number of
proteins are sequenced and compared with their known gene sequences,
methionine (or formylmethionine) occurs at the N-terminus of all of them.
However, not all amino acids are equally likely to occur second in the chain,
and the second amino acid influences whether the initial methionine is
enzymatically removed. For example, many proteins begin with methionine
followed by alanine. In both prokaryotes and eukaryotes, these proteins have the
methionine removed, so that alanine becomes the N-terminal amino acid (Table
1). However, if the second amino acid is lysine, which is also frequently the
case, methionine is not removed (at least in the sample proteins that have been
studied thus far). These proteins therefore begin with methionine followed by
lysine (Flinta et al., 1986).
Table 1 shows the N-terminal sequences of proteins in prokaryotes and
eukaryotes, based on a sample of 170 prokaryotic and 120 eukaryotic proteins
(Flinta et al., 1986). In the table, M represents methionine, A represents alanine,
K represents lysine, S represents serine, and T represents threonine.
Table 1: N-Terminal Sequences of Proteins
N- Percent of Percent of
Terminal Prokaryotic Eukaryotic
Sequence Proteins with This Proteins with This
Sequence Sequence
MA* 28.24% 19.17%
MK** 10.59% 2.50%
MS* 9.41% 11.67%
MT* 7.65% 6.67%
* Methionine was removed in all of these proteins
** Methionine was not removed from any of these proteins
Once the initiation complex is formed on the mRNA, the large ribosomal
subunit binds to this complex, which causes the release of IFs (initiation
factors). The large subunit of the ribosome has three sites at which tRNA
molecules can bind. The A (amino acid) site is the location at which the
aminoacyl-tRNA anticodon base pairs up with the mRNA codon, ensuring that
correct amino acid is added to the growing polypeptide chain. The P
(polypeptide) site is the location at which the amino acid is transferred from its
tRNA to the growing polypeptide chain. Finally, the E (exit) site is the location
at which the "empty" tRNA sits before being released back into the cytoplasm
to bind another amino acid and repeat the process. The initiator methionine
tRNA is the only aminoacyl-tRNA that can bind in the P site of the ribosome,
and the A site is aligned with the second mRNA codon. The ribosome is thus
ready to bind the second aminoacyl-tRNA at the A site, which will be joined to
the initiator methionine by the first peptide bond (Figure 5).
Figure 5: The large ribosomal subunit binds to the small ribosomal subunit
to complete the initiation complex.
The initiator tRNA molecule, carrying the methionine amino acid that will serve
as the first amino acid of the polypeptide chain, is bound to the P site on the
ribosome. The A site is aligned with the next codon, which will be bound by the
anticodon of the next incoming tRNA.
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The Elongation Phase
Figure 6
Figure Detail
The next phase in translation is known as the elongation phase (Figure 6). First,
the ribosome moves along the mRNA in the 5'-to-3'direction, which requires the
elongation factor G, in a process called translocation. The tRNA that
corresponds to the second codon can then bind to the A site, a step that requires
elongation factors (in E. coli, these are called EF-Tu and EF-Ts), as well as
guanosine triphosphate (GTP) as an energy source for the process. Upon
binding of the tRNA-amino acid complex in the A site, GTP is cleaved to form
guanosine diphosphate (GDP), then released along with EF-Tu to be recycled
by EF-Ts for the next round.
Next, peptide bonds between the now-adjacent first and second amino acids are
formed through a peptidyl transferase activity. For many years, it was thought
that an enzyme catalyzed this step, but recent evidence indicates that the
transferase activity is a catalytic function of rRNA (Pierce, 2000). After the
peptide bond is formed, the ribosome shifts, or translocates, again, thus causing
the tRNA to occupy the E site. The tRNA is then released to the cytoplasm to
pick up another amino acid. In addition, the A site is now empty and ready to
receive the tRNA for the next codon.
This process is repeated until all the codons in the mRNA have been read by
tRNA molecules, and the amino acids attached to the tRNAs have been linked
together in the growing polypeptide chain in the appropriate order. At this point,
translation must be terminated, and the nascent protein must be released from
the mRNA and ribosome.
Termination of Translation
There are three termination codons that are employed at the end of a protein-
coding sequence in mRNA: UAA, UAG, and UGA. No tRNAs recognize these
codons. Thus, in the place of these tRNAs, one of several proteins, called
release factors, binds and facilitates release of the mRNA from the ribosome
and subsequent dissociation of the ribosome.
Comparing Eukaryotic and Prokaryotic Translation
The translation process is very similar in prokaryotes and eukaryotes. Although
different elongation, initiation, and termination factors are used, the genetic
code is generally identical. As previously noted, in bacteria, transcription and
translation take place simultaneously, and mRNAs are relatively short-lived. In
eukaryotes, however, mRNAs have highly variable half-lives, are subject to
modifications, and must exit the nucleus to be translated; these multiple steps
offer additional opportunities to regulate levels of protein production, and
thereby fine-tune gene expression.
