2. Transcription,
RNA
processing, an
d translation
are the
processes that
link DNA
sequences to
the synthesis
of a specific
polypeptide
chain.

3.  Translation is a well
conserved process among
prokaryotes and 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.

4. 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.

5.  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.

6.  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.
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icro06.swf

7. 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).

8. (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.

9. 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).

10. Rules for base pairing between
the 3rd base of the codon and
anticodon are relaxed (wobble).
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
A is possible in the wobble position can “read” 3
position of the anticodon, but it codons, and a tRNA with G
is almost never found in nature. or U in the wobble position
can read 2 codons.

11. 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.

12. 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.

13. Three initiation factors (IF 1,
2, 3) and GTP bind to the
small ribosomal subunit.
The mRNA-binding site to the
ribosome is composed of a
portion of the 16S rRNA of 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.

14. 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.

15.  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.

16. 1. Elongation begins with the
binding 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.

17.  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.

18.  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.

19. A ribosome complexes with EUKARYOTIC TRANSLATION
mRNA 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 pre-initiation
complex is formed when the
40S subunit–eIF3 complex is
bound by eIF1A and a ternary
complex of the MettRNAi
Met, eIF2, and GTP

20. 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.

21. (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,
eIF5 and eIF6 are
GTP-binding proteins,
whose bound GTP is
hydrolyzed during
translation initiation.

22. .
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), followed by a
conformational change
in the ribosome induced
by hydrolysis of GTP in
EF1-GTP (2).

23. 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.

24. 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.

25. 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.

27. (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

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mations/translation/translation.htm