Cell Metabolism - Bioprocess Engineering: Kinetics, Biosystems, Sustainability and Reactor Design - page 487

points in translation. They are encoded at the end of each gene. Thus, they are called end
codons. Sometime, we also call these codons nonsense codons because they have no correspon-
dence to any amino acids.
The genetic code is essentially universal, although some exceptions exist (particularly in
the mitochondria and for inclusion of rare amino acids). This essential universality greatly
facilitates genetic engineering. The language used to make a human protein is understood
in E. coli and yeast, and these simple cells will faithfully produce the same amino acid
sequence as a human cell.
Knowing the genetic language, we may now examine the mechanism by which proteins
are actually constructed.
10.4.2. Translation: How the Machinery Works
Translation is the third stage of protein biosynthesis. In translation, mRNA produced by
transcription is decoded by the ribosome to produce a specific amino acid chain, or polypep-
tide, that will later fold into an active protein. In bacteria, translation occurs in the cell's cyto-
plasm, where the large and small subunits of the ribosome are located and bind to the
mRNA. In eukaryotes, translation occurs across the membrane of the endoplasmic reticulum
in a process called vectorial synthesis. The ribosome facilitates decoding by inducing the
binding of tRNAs with complementary anticodon sequences to that of the mRNA
(Fig. 2.35). The tRNA's carry specific amino acids that are chained together into a polypeptide
as the mRNA passes through and is “read” by the ribosome in a fashion reminiscent to that of
a stock ticker and ticker tape.
In many instances, the entire ribosome/mRNA complex will bind to the outer membrane
of the rough endoplasmic reticulum and release the nascent protein polypeptide inside for
later vesicle transport and secretion outside of the cell. Many types of transcribed RNA,
such as tRNA, rRNA, and small nuclear RNA, do not undergo translation into proteins.
The process of translation consists of four primary steps: activation, initiation, elongation,
and termination. In activation, the correct amino acid is covalently bonded to the correct
tRNA. The amino acid is joined by its carboxyl group to the C3-OH of the tRNA by an ester
bond. When the tRNA has an amino acid linked to it, it is termed “charged.” Initiation
involves the small subunit of the ribosome binding to the C5-end of mRNA with the help
of initiation factors (IFs). All protein synthesis begins with an AUG codon (or GUG) on the
m-RNA. This AUG encodes for a modified methionine, N-formylmethionine. In the middle
of a protein, AUG encodes for methionine, so the question is how the cell knows that a partic-
ular AUG is an initiation codon or for N-formylmethionine. The answer lies about six to nine
nucleotides upstream of the AUG, where the ribosome-binding site (Shine e Delgarmo box)
is located. For prokaryotes, the consensus sequence is known as Shine e Delgarmo sequence
or Shine e Delgarmo box: AGGAGG, a six base sequence just ahead of AUG. For eukaryotes,
this is known as the Kozak consensus sequence: (GCC)GCCACC AUG G or (GCC)
GCCGCC AUG G. That is, there is one base after the initiation codon AUG for eukaryotes.
Ribosome-binding sites can vary in strength and are an important consideration in genetic
engineering. The initiation of polymerization in prokaryotes requires an initiation complex
composed of a 30s ribosomal unit with an N-formylmethionine bound to its initiation region,
a 50s ribosomal unit, three proteins called IFs (PIF1, PIF2, and PIF3), and the phosphate bond