TRANSLATION

Daniel Onyejesi
INTRODUCTION
• In translation, tRNA is the adapter molecule
which recognizes the codons on mRNA and
carries the amino acid which they code to the
site of protein synthesis.
• tRNA has an anticodon
FEATURES OF tRNA
It is a single chain containing between 73 and 93
ribonucleotides
The basic feature is the structure. Its actual
structure (primary structure) is L-shaped while
its secondary structure is clover shaped i.e. the
clover leaf structure
The secondary structure of tRNA contains
several unusual bases like :
A methylated uracil
Demethylated bases
FEATURES OF tRNA contd
FEATURES OF tRNA contd
a. The TPC loop: (Thymine pseudo cytosine loop)
has a pseudo base and the number of bases
found there are variable
b. Anticodon loop: This is the portion that bears
the anticodons to the codons on the mRNA.
c. The 5՚ end: This end normally contains G
d. The 3՚ end: This normally has ACC and
the amino acid accepted by the acceptor
arm attaches to the A on the 3՚ end.
FEATURES OF tRNA contd
Anticodon on tRNA is read from 5՚-3՚ direction
i.e. the 3rd base in codon pairs with the
first base in the anticodon
WOBBLE EFFECT EXPLAINED
• Normally, the first 2 bases in codon will always pair by
watson-crick rule to the last 2 bases in the anticodon
• The last codon is generally flexible and wobbles and does
not strictly pair according to watson-crick rule and so
can produce variable amino acid
E.g. Alanyl ttRNA has the anticodon
 "IGC" and can bind to 3 codons: GCU, GCC and GCA
In the 3 codons, it is noticed that the 1st two letters( i.e.
G and C) are the same but the third differs
The third letter that is flexible is called a "wobble point"
"I" can therefore bind to U or C or A. The first base of
tRNA determines if a tRNA will have one codon or two
or three
WOBBLE EFFECT contd
• If the first base is is C or A, the tRNA can only
read one codon
• If it is U or G, the tRNA can read two codons and
when it is I, the tRNA can read 3 codons
• The amino acid that is carried by tRNA must be
activated by ATP to form an aminoacyl-tRNA
Amino acid + ATP Aminoacyl AMP
Aminoacyl AMP + ttRNA Aminoacyl-tRNA
+AMP + PPi
WOBBLE EFFECT contd
• The overall reaction is as shown/written below:
Amino Acid + ATP + tRNA Aminoacyl-
tRNA + AMP
+PPi
RIBOSOMES AND TRANSLATION
• This is a large molecule with a sedimentation
rate of 70S (i.e. E.coli ribosome). It has 2
subunits: 50S and 30S
RIBOSOMES AND TRANSLATION
contd
• 16S is the ribonucleic part/aspect of ribosome
• Ribosome has 3 sites involved in protein
synthesis and they are:
Large Subunit [50S]
Small subunit [30S]
• 50S + 30S = 70S
• The S- sedimentation coefficients or Svedberg
values are determined both by shape and
molecular mass, their numerical values are thus
not strictly additive
RIBOSOMES AND TRANSLATION
contd
• In 30S, there are 3 binding sites whose
arrangement are not straight but arranged
triangularly
• The sites are:
E- exit site
P- peptide site
A- acyl site
• Aminoacyl-tRNA synthetase is the enzyme that
catalyzes the formation of activated tRNA
RIBOSOMES AND TRANSLATION
contd
• The first amino acid to be incorporated is methionine
• The Shine Dalgarno sequence is always presen tand
recognised by the enzyme that attaches methionine
• The sequence is normally seen as UAGGAGG and
consists of 6-8 bases
• This sequence is an offspring from the 5՚-end
• The first AUG after the S-D sequence is the
initiation point, and because methionine is first to
be added, tRNA bringing methionine is formyl
tagged with methionine [i.e. f-met-tRNA].
• Other factors involved in translation exist
TRANSLATION
• There are three stages involved in translation of
mRNA:
Initiation
Elongation
Termination
INITIATION
• This is the 1st process. There are 3 initiation factors
which are: IF1, IF2, and IF3
• IF1 binds to acyl site of ribosome (A-site)
• IF3 binds to exit site of ribosome (E-site)
• After binding of IF1 and IF3, the 30S ribosome then goes
to mRNA and locates the Shine-Dalgarno sequence
• Once it binds there, it slides intil it locates the first AUG
(Met) and at AUG, the AUG is located at the P-site of 30S
subunit( i.e. the tRNA with methionine will locate and
attach to the P-site)
IF3 + 30S Complex I
IF2 + f-met-tRNA + GTP Complex II
INITIATION contd
• Complex II will bind to the initiation site at P
• The function of IF3 is not to allow 50S to bind.
• As soon as IF2 component of complex II binds to
P-site. IF3 at E-site is released and the 50S then
binds to the 30S to form a 70S complex and at
this point, initiation stops.
Complex I + Complex II Complex III
• The aim of initiation is to form a 70S complex
ELONGATION
• This involves the addition of more amino acids
to the chain
• This occcurs in 3 stages called the elongation
cycle and they include:
Aminoacyl-tRNA binding
Peptide bond formation
Translocation
AMINOACYL-tRNA BINDING
• This involves the binding of the next activated
tRNA cattying the next amino acid
• This tRNA will bind to the A-site
• However this cannot be done without the help of
an enzyme
• Hence the tRNA will form a complex with Ef-Tu
and GTP
tRNA + Ef-Tu + GTP Complex
• Ef-Tu + GTP will be hydrolysed to Ef-Tu + GDP
+ Pi
Aminoacyl-tRNA binding contd
• This is done by another complex, Ef-Ts which
will cause the release of GDP and Ef-Tu
PEPTIDE BOND FORMATION
• This is cayalysed by peptidyl transferase which
detaches the Amino acid from the P-site at the
carboxyl end and transfers it to the Amino Acid
on the tRNA on A site
• The carboxyl end of the detached Amino acid is
attached to the NH2-end of the Amino Acid on
the A-site
• The function of the enzyme resides on the 16S
component of 30S subunit
TRANSLOCATION
• This happens only in the presence of Ef-G
(Translocase) which requires an eneegy source, so
we have Ef-G-GTP complex
• Three (3) things occur simultaenously:
1. The deacylated tRNA on the P-site will move to the
E-site.
2. The tRNA carrying the growing chain will move to
the P-site leaving the A-site vacant
3. Ribosome moves/shifts on mRNA to position the
A site on the next codon and the deacylated tRNA
on E-site will be released before another/the next
tRNA carrying another amino acid on the A-site.
TERMINATION
• This entire process conyinues until a
nonsense/stop/termination codon is met and
because no tRNA has anticodon for the nonsense
codon, the process is terminated.
• The last amino acid on the growing chain will have
its end
• Release factors will be required by the enzyme(Rf 1-
3)
• Rf1 will recognize UAG and UAA and Rf2 will
recognize UGA
• RF3 assists Rf1 and Rf2 by hydrolysing tRNA.