Download Translation - Genes to proteins

January 10, 2010
[TRANSLATION - GENES TO PROTEINS]
Transcription gets the genetic information from the DNA out into the cytoplasm where new proteins are
synthesized, but how is the information in the mRNA molecule used to direct the assembly of amino
acids into a protein? The primary structure of a protein is the number and order of amino acids; there
are 20 amino acids that can be found in proteins, but there are only four nitrogenous bases used in RNA.
How do the four bases of RNA specify twenty amino acids? And, how are proteins started and stopped?
A simple math test can provide a clue about the nature of the genetic code. If you have 4 bases and
combinations of 2 of them are required to specify an amino acid, then 4 squared is 16, but there are 20
amino acids. So, could they each be specified by a combination of three bases (4 cubed = 64). The sixty
four possibilities created by 3-base “words” would clearly allow the code to specify all of the required
amino acids? But what about all of the “extra” combinations? Are they just not used, or is the code
redundant, with more than one 3-base word specifying the same amino acid?
How does the code work? What are the start and stop signals that mark the beginning and end of a
polypeptide chain? Is the code overlapping, with the 3-base words running on top of each other, or is
the RNA message read 3 bases at a time?
Francis Crick and colleagues published a paper in Nature in 1961 that set out arguments for a
nonoverlapping, 3-base code. Their argument that the code is nonoverlapping is based on work by other
groups (Wittmann; Tsugita and Fraenkel-Conrat) on tobacco mosaic virus. These groups showed that in
TMV when the RNA is treated with nitrous acid, usually only one amino acid at a time is altered. If the
code was overlapping, they would have predicted that three amino acids would be changed with each
single mutation. Additionally work on human hemoglobin showed that mutations changed only one
amino acid at a time.
In the experimental work described in this paper, Crick induced deletion or insertion mutations in the rII
gene of bacteriophage T4. Single-base insertions or deletions should produce a shift in the “reading
frame” of the triplet code. His group was able to show that a single mutation gave a mutant phenotype,
but a combination of a single base addition and a single base deletion near to one another produced a
normal phenotype. They also later showed that 2-base deletions or insertions conferred mutant
phenotypes but that 3-base insertions or deletions were almost always wildtype.
Translation, or protein synthesis, is directed in eukaryotic cells by an mRNA molecule. Translation can be
seen to occur in two phases: (1) information transfer, in which RNA base sequence of the mRNA
determines the sequence of amino acids and (2) chemical processes, in which the peptide bonds
between the adjacent amino acids are formed. The components required for translation include: mRNA,
ribosomes, tRNA, aminoacyl tRNA synthetases, and accessory proteins involved in initiation, elongation
and termination.
Ribosomes consist of two subunits that are named based on their size. Each of these subunits contains
ribosomal RNA and protein. Ribosome subunit size is determined by their sedimentation rate an is
indicated by S (Svedberg unit). In bacteria the large ribosome subunit is 50S, and the small is 30S. In
eukaryotes the large subunit is 60S, and the small is 40S.
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Dr Gihan Gawish
January 10, 2010
[TRANSLATION - GENES TO PROTEINS]
The events translation initiation are the binding of the small ribosomal subunit to the mRNA molecule
and the binding of the charged tRNA that bears the first amino acid of the polypeptide chain. The
binding of the small ribosomal subunit is mediated by the 16S RNA that is part of the small ribosomal
subunit in prokaryotes. In eukaryotes the 5´ methyl cap of the mRNA is involved in binding the small
ribosomal subunit. The small ribosomal subunit, mRNA and tRNA complex recruits the large ribosomal
subunit.
Elongation can be thought to involve three processes: 1) aligning each aminoacylated tRNA, 2) forming
the peptide bond to add the new amino acid to the polypeptide chain, and 3) moving the ribosome
along the mRNA by three more bases (one codon).
EF-Tu (prokaryotes) or EF-1alpha (eukaryotes) aligns each new tRNA into the A site of the large subunit.
This process requires the hydrolysis of GTP. When the A site is filled, a peptidyl transferase activity
catalyzes the formation of the peptide bond between the amino acid in the A site and the adjacent
amino acid in the P site. The formation of the peptide bond requires several proteins and RNA molecules
of the large subunit and some evidence suggests that the peptide bond formation is catalyzed by an RNA
molecule.
After the peptide bond is formed the ribosome translocates 3 bases, the tRNA from the P-site that is no
longer attached to its amino acid moves to the E site, and the tRNA with amino acid from the A site is
now occupies the P site. The A site is now empty. This is known as the “posttranslocation state” for the
ribosome.
A charged tRNA bearing the next amino acid in the polypeptide chain binds to the codon of the mRNA
and occupies the A site. The uncharged tRNA in the E site is released. This is known as the
posttranslocation state.
This process is repeated, with the ribosome reading the mRNA molecule one codon at a time.
Elongation proceeds until a stop codon is reached. There are three stop codons in the genetic code:
UAG, UGA, UAA. There is no tRNA that bears an anticodon that can read any of the stop codons (the
anticodons would be AUC, ACU, or AUU). Therefore no tRNA can fill the empty A site of the elongdation
complex. Instead the A site is filled by a release factor, and the amino acid is cleaved from the tRNA that
occupies the P site of the ribosome.
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Dr Gihan Gawish