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Translation
Slide 2
Two distinct stages are required to synthesize a protein. As we saw in the
previous lesson, the first stage is transcription which involves three major steps
(initiation, elongation and termination) to transcribe the information from genes
contained within a DNA molecule to produce different types of RNA. The second stage
is translation. Once again, three steps are required to interpret the genetic information
contained within a mRNA molecule to produce a polypeptide. The focus of this lesson is
on the process of translation.
Slide 3
Recall that transcription takes place where you find the DNA, i.e. the
cytoplasm of prokaryotic cells and the nucleus of eukaryotic cells. Translation on the
other hand, occurs on ribosomes in the cytoplasm of both prokaryotic and eukaryotic
cells.
Slide 4
How is the genetic material encoded in mRNA read? The linear sequence
of nucleotides is read three bases at a time. The triplets of bases on mRNA are called
codons. In this table you can count 64 codons (4 x 4 x 4) that code for 20 different amino
acids. The genetic code is said to be universal since it applies to ALL organisms. Note
that most amino acids are encoded by more than one codon. However, every codon
always encodes for only one amino acid; thus the genetic code is unambiguous. The first
two letters for the codons of a particular amino acid are always the same, the third letter
may vary. For example, look at glycine (bottom right corner); the first two letters are
GG, the third letter may be U, C, A or G. This allows some flexibility in codonanticodon pairing between the amino acid and transfer RNA. This flexibility is called the
“wobble effect”. As is often the case, there is an exception to the rule; in this case it
would be the amino acid leucine where only the second letter is the same. Start and stop
codons indicate where translation begins and ends. The amino acid methionine,
determined by the codon AUG, is always the start codon initiating translation. Stop
codons that end translation are determined by the base triplets UAA, UAG or UGA.
Slide 5
As we saw in our discussion of transcription, the order of bases on mRNA
is determined by the order of the bases on the DNA template strand. Construction of
mRNA is according to base pair rules: A codes for U, T codes for A, C codes for G and G
codes for C. During translation, the bases on the mRNA strand are read as triplets
(codons), each triplet coding for a particular amino acid. Peptide bonds are formed to
join amino acids together and form a polypeptide.
Slide 6
The function of transfer RNA is to transfer free amino acids from the
cytoplasm to a ribosome so they can be placed in the proper order according to the
sequence of bases in the mRNA. This figure demonstrates three different ways to
represent a transfer RNA molecule. Transfer RNA is a small molecule that base pairs
with itself giving the molecule a characteristic “t” shape. The bases of the anticodon pair
with the correct codon of mRNA, thereby introducing amino acids in the order that they
are to be incorporated into the polypeptide. Note that the anticodon region of the
cloverleaf model has three bases; these are unique to each tRNA type. Amino acids are
always attached to the 3´ site opposite from the anticodon region. The cell cytoplasm is
full of transfer RNA molecules and free amino acids waiting to be assembled into
polypeptides.
Slide 7
If the cytoplasm is full of free amino acids and transfer RNA molecules
how does the correct amino acid match up with the appropriate tRNA? This is
accomplished by the enzyme aminoacyl-tRNA synthetase; a specific version of this
enzyme is present in the cytoplasm for each amino acid. As indicated in the diagram, this
enzyme has an amino acid site, an ATP site and a tRNA site. The enzyme catalyzes a
reaction between ATP and the correct amino acid thus activating the amino acid. The
appropriate tRNA can now be accepted by the enzyme and the tRNA and amino acid are
covalently bound together. The enzyme releases the aminoacyl tRNA which is now
ready to transport a specific amino acid to the site of polypeptide assembly, the ribosome.
Slide 8
Before we can discuss the process of translation, we first need to look
more closely at the ribosome. A ribosome is made up of a large subunit and a small
subunit which only come together during translation. When the ribosome is not
interpreting mRNA the subunits remain separate in the cytoplasm. Each subunit is
composed of ribosomal RNA and proteins. Ribosome subunits are produced in the
nucleolus of eukaryotic cells and then exported to cytoplasm. A ribosome is not
associated with just one type of protein; it can transcribe many different mRNA
molecules making many different polypeptides.
The genetic information on the mRNA is translated at specific sites on the ribosome.
There are four docking sites associated with the large subunit: T, A, P, E. The transfer
(T) site is where the activated amino acid arrives at the ribosome accompanied by a
“transfer factor”. The amino acid (A) site is where the tRNA anticodon pairs with the
mRNA codon holding the next amino acid to be added to the chain. At the polypeptide
(P) site the tRNA holds the amino acid that is being incorporated into the polypeptide
chain. Once the amino acid is incorporated into the growing polypeptide chain, the tRNA
is discharged through the exit (E) site. Note that the tRNA and mRNA only interact at
the A and P sites on the ribosome.
Slide 9
The synthesis of a polypeptide can be divided into three steps. The first
step, initiation, occurs when the ribosome subunits and mRNA come together;
elongation is the second step and occurs when amino acids are added to the growing
polypeptide chain; termination is the final step when a stop codon ends translation of the
polypeptide and it is released from the ribosome. Now let’s look at the events that occur
during each stage.
Slide 10
The small subunit of the ribosome recognizes a specific sequence on the
mRNA that is adjacent to the start codon and binds to it. The tRNA with the first amino
acid of the polypeptide matches up with the start codon AUG. The first amino acid is
always methionine. The large subunit of the ribosome joins the initiation complex. Once
this has occurred the active tRNA is docked in the P site of the large subunit. The A site
is now ready to accept the next tRNA and begin building the polypeptide.
Slide 11
The polypeptide chain becomes longer, one amino acid at a time. The
next active tRNA docks in the A site. In this example it carries the amino acid proline.
The large ribosome subunit catalyzes the formation of a peptide bond between the two
amino acids. The free tRNA exits the ribosome complex and the ribosome shifts over
one codon. The tRNA with proline and methionine now occupies the P site leaving the A
site vacant. Another aminoacyl tRNA with an anticodon matching the mRNA docks in
the A site. The amino acid is added to the growing polypeptide through formation of
another peptide bond. The ribosome again shifts one codon and the tRNA exits from the
E site. This continues until the polypeptide reaches its designated length. Notice that
during translation the 5´ end of the mRNA moves through the ribosome first. How long
does it take to make a polypeptide chain, you ask? Ten or more amino acids may be
added per second! Considering the mechanics and biochemical reactions that are
involved, the process of translation is very rapid.
Slide 12
The growing polypeptide is complete once a stop codon in the mRNA –
either UAA, UAG or UGA - enters the A site. A protein release factor binds to the stop
codon in the A site and a water molecule is added to the polypeptide in place of an amino
acid causing it to be released from the ribosome. All the components associated with the
ribosome disassemble and return to the cytoplasm as individual components.
Slide 13
Once the newly formed polypeptide is released from a ribosome it folds
into a three dimensional shape depending on the amino acid sequence of the protein.
Towards the end of translation signal peptides are incorporated into the molecule. These
signal peptides provide a specific “instruction” that determines if the polypeptide is to
complete translation in the endoplasmic reticulum, or translation is to be completed in the
cytoplasm. If completed in the cytoplasm, signal peptides direct the protein to specific
organelles in the cell.
Slide 14
In order to accommodate the high turnover of proteins in a cell, many
polypeptides can be synthesized from the same strand of mRNA at the same time. This
chain of actively translating ribosomes is called a polysome or polyribosome.