Download Lecture 6 Translation

Chapter 6
Gene Expression:
Translation
Translation
• Nucleotides specify the amino-acid
sequence in proteins with four different
nucleotides. A, C, G and U.
• A three letter code (using three of the four
nucleotides) generates 64 possible
combinations or CODONS.
• These 64 combinations are more than
enough to codify for the 20 amino acids
that forms proteins.
• Evidence for a triplet code came from
experiments performed in the early 1960’s by
Crick, Barner, Brennet and Watts-Tobin in
bacteriophage T4.
• The exact relationship of the 64 codons to
the 20 Amino Acids was determined by
Nirenberg and Khorana in 1968.
• Based on Nirenberg and Khorana’s work, we
know that by convention, a codon is written
as it appears in mRNA, reading in the 5’ to 3’
direction.
Characteristics of the Genetic
Code
• a. It is a triplet code. Each three-nucleotide
codon in the mRNA specifies one amino in the
polypeptide.
• b. It is comma free. The mRNA is read
continuously, three bases at a time, without
skipping any bases.
• c. It is non-overlapping. Each nucleotide is part
of only one codon, and is read only once during
translation.
• d. It is almost universal. In nearly all organisms
studied, most codons have the same amino acid
meaning.
• e. It is degenerate. Of 20 amino acids, 18 are encoded
by more than one codon. Met (AUG) and Trp (UGG) are
the exceptions; all other amino acids correspond to a set
of two or more codons. Codon sets often show a pattern
in their sequences; variation at the third position is most
common.
• f. The code has start and stop signals. AUG is the
usual start signal for protein synthesis and defines the
open reading frame. Stop signals are codons with no
corresponding tRNA, the nonsense or chain-terminating
codons. There are generally three stop codons: UAG
(amber), UAA (ochre), and UGA (opal).
• g. If each anticodon had to be a perfect match
to each codon, we would expect to find 61 types
of tRNA, but the actual number is about 45. This
is because the anticodons of some tRNAs
recognize more than one codon.
• This is possible because the rules for base
pairing between the third base of the codon and
anticodon are relaxed (called wobble). At the
wobble position, U on the anticodon can bind
with A or G in the third position of a codon.
• Some tRNA anticodons include a modified form
of adenine, inosine, which can hydrogen bond
with U, C, or A on the codon.
Translation: The Process of Protein
Synthesis
• Protein synthesis take places in the ribosomes where the
genetic message encoded in mRNA is decoded and translated.
• The mRNA is translated 5’-to-3’, and the polypeptide is made
in the N-terminal- to- C-terminal direction.
• Amino acids are brought to the ribosomes bound to tRNAs and
the amino acids are inserted in the proper sequence due to:
– The specific binding of each amino acid to its tRNA.
– The specific base-pairing between the mRNA codon and
complementary tRNA anticodon.
The tRNA
All tRNAs can be shown in a cloverleaf structure, with
complementary base pairing between regions to form four
stems and loops (Figure 5.20). Loop II contains the anticodon
used to recognize mRNA codons during translation. Folded
tRNAs resemble an upside-down “L.”
Details
•1. How does the tRNA add an amino acid?
The amino acid is attached to the tRNA by the
enzyme aminoacyl tRNA synthetase. The
process is called amino acetylation and the tRNA
with an amino acid is called “charged tRNA”.
Details
• 2. How does the mRNA recognizes the
anticodon?
The mRNA recognizes the anticodon but
not the amino acid carried by the tRNA.
Translation
• 1. Protein synthesis is similar in prokaryotes and eukaryotes.
Some significant differences do occur and we will mention as
we go.
• 2.
a.
b.
c.
In both it is divided into three stages:
Initiation.
Elongation.
Termination.
• In prokaryotes initiation of translation requires: A mRNA, a
ribosome, a specific initiator tRNA, initiation factors, GTP for
energy and Mg2+ (magnesium ions).
Initiation
• In prokaryotes the first step is the boinding
of the small ribosome subunit (30s) to the
mRNA region where the AUG (start
codon) is located.
1. The 30S ribosomal subunit binds to
IF1, IF2, IF3, GTP and Mg ions.
• The mRNA has a sequence upstream (5’)
of the start codon AUG (about 8-12 nt)
known as the ribosome binding site or
RBS. It is a purine (AG) rich area. The
RBS is also known as the Shine-Dalgano
sequence.
„The
recognition of the complementary base pairs
between the RBS of the mRNA and the 16S
ribosomal RNA of the small ribosome subunit
allows the ribosome to locate the precise location
for the initiation of protein synthesis.
• Next, the initiator tRNA binds the AUG to
which the 30S subunit is bound. AUG
universally encodes methionine. Newly made
proteins begin with Met, which is often
subsequently removed.
• In prokaryotes, initiator
methionine is formylmethionine
(fMet). It is carried by a specific
tRNA (with the anticodon 5’CAU-3’). It is a methionine
with a formil group. The rests
of methionines in the chain are
added by normal met tRNA.
• When fMet-tRNA binds to the start codon of
the 30S-mRNA complex, IF3 is released and the
50S ribosomal subunit binds the complex. GTP
is hydrolyzed, and IF1 and IF2 are released. The
result is a 70S initiation complex consisting of:
• a. mRNA.
• b. 70S ribosome (30S and
50S subunits) with a vacant
A site.
• c. fMet-tRNA in the
ribosome’s P site.
• The main differences in eukaryotic translation are:
• a. Initiator methionine is not modified. As in
prokaryotes, it is attached to a special tRNA.
• b. Ribosome binding involves the 5’ cap, rather than a
Shine-Delgarno sequence and a large group of initiator
factors.
• c. The eukaryotic mRNA’s 3’ polyA tail also plays a
role in initiation. The PolyA binding protein (PABP)
that is bound to the polyA tail binds to the initiator
factor bound to the cap, thus loping the 3’ of the
mRNA close to the 5’ end. The PoliA tail stimulates
the inititation of translation.
Elongation of the Polypeptide
Chain
• Elongation of the amino acid chain has
three steps (Figure 6.13):
• a. Binding of aminoacyl-tRNA to the
ribosome.
• b. Formation of a peptide bond.
• c. Translocation of the ribosome to the
next codon.
REVIEW
• Each ribosome has a binding site for
mRNA and three binding sites for tRNA
molecules.
•
–
–
–
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.
Binding of Aminoacyl-tRNA
• At the start of elongation the anticodon of fMet
is bonded the AUG initiation codon (hydrogen
bonds) in the P site . The next codon in th
mRNA is the A site.
• The EF-Tu is called protein elongation factor.
• The next charged tRNA approaches the
ribosome bound to EF-Tu-GTP. When the
charged tRNA hydrogen bonds with the codon
in the ribosome’s A site, hydrolysis of GTP
releases EF-Tu-GDP.
EF-Tu is recycled with assistance from EF-Ts, which
removes the GDP and replaces it with GTP, preparing
EF-Tu-GTP to escort another aminoacyl tRNA to the
ribosome.
Peptide Bond Formation
• 1. The two aminoacyl-tRNAs are positioned by the
ribosome for peptide bond formation, which occurs in
two steps (Figure 6.14):
• a. In the P site, the bond between the amino acid and
its tRNA is cleaved.
• b. Peptidyl transferase forms a peptide bond between
the now-free amino acid in the P site and the amino
acid attached to the tRNA in the A site.
• c. The tRNA in the A site now has the growing
polypeptide chain attached to it.
Translocation
• 1. The ribosome now advances one codon
along the mRNA. EF-G (elongation factor G )
is used in translocation in prokaryotes. EF-GGTP binds the ribosome, GTP is hydrolyzed,
and the ribosome moves one codon while the
uncharged tRNA leaves the P site. (Eukaryotes
use a similar process, with a factor called eEF-2.)
• 2. During translocation the peptidyl-tRNA remains
attached to its codon, but is transferred from the
ribosomal A site to the P site by an unknown
mechanism.
• 3. Release of the uncharged
tRNA involves the 50S
ribosomal E (for Exit) site.
Binding of a charged tRNA
in the A site is blocked until
the spent tRNA is released
from the E site.
• 4. The vacant A site now contains a new codon, and
an aminoacyl-tRNA with the correct anticodon can enter
and bind. The process repeats until a stop codon is
reached.
• 5. Elongation and translocation are similar in
eukaryotes, except for differences in number and type of
elongation factors and the exact sequence of events.
• 6. In both prokaryotes and eukaryotes, simultaneous
translation occurs: New ribosomes may initiate as soon
as the previous ribosome has moved away from the
initiation site, creating a polyribosome (polysome); an
average mRNA might have 8–10 ribosomes (Figure
6.15).
Termination
• Termination is signaled by a stop codon
(UAA,UAG,UGA), which has no corresponding
tRNA (Figure 6.16).
• Release factors (RF) assist the ribosome in
recognizing the stop codon and terminating
translation. In E. coli (RF1, RF2 and RF3).
• In eukaryotes, eRF is the only termination
factor, recognizing all three stop codons and
stimulating termination.
Sequence of termination events
1. Stop codon is encountered
and there is not amynoacyltRNA that corresponds with a
stop codon.
2. A release factor binds to the
stop codon.
3. The polypeptide chain is
realease from the tRNA in the
A site.
4. All the components separate.
5. Methionine or fMet are cleaved
and removed.
Protein sorting
How does the protein know where to go? Is
it a protein that is going to go to an organelle
or is it going to be secreted?
In eukaryotes, proteins synthesized on the
rough ER (endoplasmic reticulum) are
glycosylated (signaled) and then transported
in vesicles to the Golgi apparatus. The Golgi
sorts proteins based on their signals, and
sends them to their destinations.