TRANSLATION

Mrs. Ofelia Solano

Saludar
Department of Natural Sciences
University of St. La Salle
 Transcription,
RNA processing,
and translation are
the processes that
link DNA
sequences to the
synthesis of a
specific
polypeptide chain.
 Translation is a well conserved
process among prokaryotes and
TRANSLATION
eukaryotes.
 Ribosomes catalyze the joining of
the amino acid monomers directed
by the mRNA sequence.
 Amino-acyl tRNA synthetases
attach amino acids to the
appropriate tRNAs.
 The amino-acyl tRNA act as
adaptors in the translation of the
nucleic acid sequence of the mRNA
into the amino acid sequence of the
protein.
 Additional processing and
assembly is often required to
modify the proteins.
 THE TOOLS OF TRANSLATION
1. Ribosomes
 Each ribosome has a large and a small subunit.
 These are composed of proteins and rRNA, the most abundant
RNA in the cell.
 rRNA is the main constituent at the interphase between the two
subunits and of the A and P sites.
 It is the catalyst for peptide bond formation.
 After rRNA genes are transcribed to rRNA in the nucleus, the
rRNA and proteins form the subunits in the nucleolus. The
subunits exit the nucleus via nuclear pores.
 The large and small subunits join to form a functional
ribosome only when they attach to an mRNA molecule.
 A functional ribosome has A (aminoacyl) and P (peptidyl) sites
as cavities on the ribosome where charged tRNA (carrying an
amino acid) molecules bind during polypeptide synthesis.
 The recently postulated E (exit) site is the site from which
discharged tRNAs leave the ribosome.
 The mRNA-binding site binds a sequence near the 5’ end of the
mRNA, placing
the mRNA in
the proper position
for the translation of
its first codon.
 The binding sites are
located at or near the
interface between the
large and small subunits.
 The P site holds the
tRNA carrying the
growing
polypeptide chain.
 The A site carries
the tRNA with the
next amino acid.
 Discharged tRNAs
leave the ribosome
at the E site.
2. tRNA molecules consists of a strand of about 80
nucleotides that folds back on itself to form a 3D structure.
It contains:
1) three major loops,
2) four base-paired regions,
3) an anticodon triplet and
4) a 3’ prime terminal sequence of CCA (where the
appropriate amino acid can be attached by an ester bond).
 During maturation of the tRNA molecule a number of
nucleotides are modified in tRNA specific ways.
 The modified nucleotides in the tRNA structure are inosine
(I), methylinosine (mI), dihydrouridine (D), ribothymidine
(T), pseudouridine (¥) and methylguanosine (Gm).
 Structure of tRNAs. (a) Although the exact nucleotide sequence varies among tRNAs,
they all fold into 4 base-paired stems and 3 loops. The CCA
sequence at the 3 end also is found in all tRNAs. Attachment of an amino acid to the 3’ A
yields an aminoacyl-tRNA. Dihydrouridine (D) is present in the D loop; ribothymidine (T)
and pseudouridine (Ý) are in the TCG loop. The triplet at the tip of the anti-codon loop
base-pairs with the corresponding codon in mRNA. (b) 3-D model of the generalized
backbone of all tRNAs.
3.Twenty different aminoacyl-tRNA synthetases link
amino acids to the correct tRNAs.
 Some recognize only one tRNA, some recognize a few
because of the redundancy in the genetic code.
 Although there are 61 possible codons, there are far
fewer tRNAs.
 A number of codons that encode the same amino acid
differ only in the third position of the codon.
 A slight shift or "wobble" in the position of the base
guanine in a tRNA anticodon would permit it to pair with
uracil instead of its normal complementary base
(cytosine).
Rules for base pairing between the 3rd Nonstandard codon-anticodon
base of the codon and anticodon are base pairing at the wobble
relaxed (wobble). position.
The base in the 3rd (or wobble)
position of an mRNA codon often
forms a nonstandard base pair
with the base in the 1st position of
a tRNA anticodon. Wobble
pairing allows a tRNA to
recognize more than one mRNA
codon (top); it allows a codon to
be recognized by more than one
kind of tRNA (bottom), although
each tRNA will bear the same
amino acid. A tRNA with I
(inosine) in the wobble position
can “read” 3 codons, and a tRNA
with G or U in the wobble
position can read 2 codons.
A is possible in the wobble position of
the anticodon, but it is almost never
found in nature.
 Aminoacyl-tRNA synthetases catalyzes the formation of an ester bond between the
carboxyl group of an amino acid and the 3’ OH group of the appropriate tRNA. The amino
acid and a molecule of ATP enter the active site of the enzyme. The ATP loses
pyrophosphate and the resulting AMP bonds covalently to the amino acid. The
pyrophosphate is hydrolyzed into two phosphate groups. The tRNA covalently bonds to the
amino acid to displace the AMP and the aminoacyl tRNA is then released from the enzyme.
 THE PROCESS OF TRANSLATION

All 3 phase of translation (initiation, elongation, termination) require protein “factors” that aid
in the translation process. Both initiation and chain elongation require energy provided by the
hydrolysis of GTP.
 Formation of the prokaryotic
translation initiation complex

Three initiation factors (IF 1, 2, 3) and

GTP bind to the small ribosomal
subunit.

The 3’ end of the 16S rRNA bears a

pyrimidine-rich stretch that base pairs
with the Shine-Dalgarno sequence of
the mRNA.

The initiator aminoacyl tRNA and mRNA

are attached. The mRNA-binding site is
composed of a portion of the 16S rRNA of
the small ribosomal subunit.
 The large ribosomal subunit
joins the complex.

The resulting 70S initiation

complex has fMet-tRNA-fMet
(N-formyl-methionine)
residing in the ribosome's P
site.
 The Shine- Dalgarno sequence in E. coli is AGGAGGU, which
helps recruit the ribosome to the mRNA to initiate protein
synthesis by aligning it with the start codon.
 The complementary sequence (UCCUCC) is located at the 3' end
of the 16S rRNA in the ribosome.
 Mutations in the Shine-Dalgarno sequence can reduce translation.
This reduction is due to a reduced mRNA-ribosome pairing
efficiency, as evidenced by the fact that complementary mutations
in the anti-Shine-Dalgarno sequence can restore translation.
 When the Shine-Dalgarno sequence and the anti-Shine-Dalgarno
sequence pair, the translation initiation factors IF2-GTP, IF1, IF3,
as well as the initiator tRNA fMet-tRNA(fmet) are recruited to the
ribosome.
 The eukaryotic equivalent of the Shine-Dalgarno sequence is
called the Kozak sequence.
 1. Elongation begins with the binding
ELONGATION of the 2nd aminoacyl tRNA at the A
site. The tRNA is escorted to the A
site by the elongation factor EF-
Tu, which also carries two bound
GTPs. As the tRNA binds, the
GTPs are hydrolyzed and EF-Tu is
released. EF-Ts help recycle the
EF-Tu.
2. A peptide bond is formed between
the carboxyl group of the terminal
amino acid at the P site and the
amino group of the newly arrived
amino acid at the A site. This
reaction is catalyzed by the
peptidyl transferase activity of the
23S rRNA molecule in the large
ribosomal subunit.
3. After EF-G-GTP binds to the
ribosome and GTP is hydrolyzed,
the tRNA carrying the elongated
polypeptide translocates from the
A site to the P site. The discharged
tRNA moves from the P site to the
E (exit) site and leaves the
ribosome. As the peptidyl tRNA
translocates, it takes the mRNA
along with it. The next mRNA
codon is moved into the A site,
which is open for the next
aminoacyl tRNA.
4. These events are repeated for each
additional amino acid.
  Termination of protein synthesis
depends on release factors that
recognize the 3 stop codons.
 When a stop arrives at the A site,
it is recognized and bound by a
protein release factor (RF1 =
UAA or UAG
RF2 = UAA or UGA
RF3 = a GTPase like EF-Tu and
binds in a similar A-site
location).
 This RF causes the poly-peptide
to be transferred to a molecule of
H2O to cause its release from the
tRNA and the dissociation of the
other components of the
elongation complex.

TERMINATION
 Typically a single mRNA is used to make many copies of
a polypeptide simultaneously.
 Multiple ribosomes, polyribosomes, may trail along the
same mRNA.
 A ribosome requires less than a minute to translate an
average-sized mRNA into a polypeptide.
A ribosome complexes with mRNA EUKARYOTIC TRANSLATION
and an activated initiator tRNA, at
the start codon. The Kozak
consensus sequence,
gccRccAUGG, (R is a purine 3
bases upstream of the AUG), is
recognized by the ribosome as the
translational start site. Large and
small ribosomal subunits not
actively engaged in translation are
kept apart by binding of 2 initiation
factors, designated eIF3 and eIF6
in eukaryotes. A translation
preinitiation complex is formed
when the 40S subunit–eIF3 complex
is bound by eIF1A and a ternary
complex of the MettRNAi Met,
eIF2, and GTP
 Initiation of translation in
eukaryotes. When a ribosome
dissociates at the termination of
translation, the 40S and 60S
subunits associate with initiation
factors eIF3 and eIF6, forming
complexes that can initiate
another round of translation.
(1) and (2) Sequential addition
of the indicated components to
the 40S subunit–eIF3 complex
forms the initiation complex.
(3) Scanning of the mRNA
by the associated initiation
complex leads to
positioning of the small
subunit and bound Met-
tRNAi Met at the start
codon.
(4) Association of the
large subunit (60S) forms
an 80S ribosome ready to
translate the mRNA. Two
initiation factors, eIF2 and
eIF5 are GTP-binding
proteins, whose bound
GTP is hydrolyzed during
translation initiation.
 Cycle of peptidyl chain
elongation during translation
in eukaryotes.
Once the 80S ribosome with
Met-tRNAi Met in the
ribosome P site is assembled
(top), a
ternary complex bearing the
2nd amino acid (aa2) coded by
the mRNA binds to the A site
(1). Following a
conformational change in the
ribosome induced by
hydrolysis of GTP in EF1-
GTP (2).
 The large rRNA catalyzes peptide bond
formation between Meti and aa2 (3).
Hydrolysis of GTP in
EF2-GTP causes change in the ribosome
that results in its translocation one codon
along the mRNA and shifts the
unacylated tRNAi -Met to the E site and
the tRNA with the bound peptide to the P
site (4). The cycle can begin again with
binding of a ternary complex bearing aa3
to the now-open A site. In the 2nd and
subsequent elongation cycles, the tRNA
at the E site is ejected during (2) as a
result of the conformational change
induced by hydrolysis of GTP in EF1-
GTP.
 Termination of translation in
eukaryotes.
When a ribosome bearing a nascent
protein chain reaches a stop codon
(UAA, UGA, UAG), release factor
eRF1 enters the ribosomal complex,
probably at or near the A site
together with eRF3-GTP. Hydrolysis
of the bound GTP is accompanied by
cleavage of the peptide chain from
the tRNA in the P site and
release of the tRNAs and the two
ribosomal subunits.
 Model of protein synthesis on circular polysomes and recycling of ribosomal
subunits. Multiple individual ribosomes can simultaneously translate a eukaryotic
mRNA, shown here in circular form stabilized by interactions between proteins bound at
the 3’ and 5’ ends. When a ribosome completes translation and dissociates from the 3’
end, the separated subunits can rapidly find the nearby 5’ cap (m7G) and initiate another
round of synthesis.
 Overview of protein structure
and function.
(a) The linear sequence of amino
acids (10 structure) folds into
helices or sheets (20 structure)
which pack into a globular or
fibrous domain (30 structure).
Some individual proteins self-
associate into complexes (40
structure).
(b) Proteins display functions that
arise from specific binding
interactions and conformational
changes in the structure of a
properly folded protein.

PROTEIN STRUCTURE, TARGETING AND SORTING.pptx

 http://highered.mcgraw-hill.com/olc/dl/120077/bio25.swf

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http://www.wiley.com/college/boyer/0470003790/animations/tr
anslation/translation.htm