Download Diapositiva 1 - Universidad Autónoma de San Luis Potosí

Prokaryote
Translation
CA García Sepúlveda MD PhD
Laboratorio de Genómica Viral y Humana
Facultad de Medicina, Universidad Autónoma de San Luis
Potosí
1
Introduction
• Ribosomes provide the environment for controlling the interaction
between mRNA and aminoacyl-tRNA.
• Behave like a small migrating factories travelling along the mRNA
template engaging in rapid cycles of peptide bond synthesis.
• Possess several active centers, each constructed from a particular
group of proteins associated with rRNA.
• The active centers require rRNA for structural and catalytic roles.
• Some catalytic functions require particular proteins, but none of the
activities can be reproduced by isolated proteins or groups of proteins;
they function only in the context of the ribosome (associated with
RNA).
2
Molecular Proportions
• Each ribosome subunit has specific roles.
• mRNA is associated with the small subunit (~30 bases
of the mRNA).
• tRNAs are quite large relative to the ribosome; they
become inserted into internal sites that stretch across
the subunits.
• A third tRNA may remain present on the ribosome
after it has been used in protein synthesis, before
being recycled (prokaryotes only).
• Polypeptide elongation involves reactions at just two of
the ~10 codons covered by the ribosome.
3
Ribosomal organisation
• Each tRNA lies in a distinct site on the ribosome, the
two sites have different features:
– Incoming aminoacyl-tRNA binds to the codon
exposed on the A site (or acceptor site).
4
Ribosomal organisation
• Each tRNA lies in a distinct site on the ribosome, the
two sites have different features:
– A salient peptidyl-tRNA (tRNA carrying the
polypeptide) occupies the codon in the P site (or
donor site).
5
Ribosomal organisation
• Each tRNA lies in a distinct site on the ribosome, the
two sites have different features:
– The end of the tRNA that carries an amino acid is
located on the large subunit.
6
Ribosomal organisation
• Each tRNA lies in a distinct site on the ribosome, the
two sites have different features:
– The anticodon at the other end interacts with the
mRNA bound by the small subunit.
– So the P and A sites each extend across both
ribosomal subunits.
7
Ribosomal organisation
• Each tRNA lies in a distinct site on the ribosome, the
two sites have different features:
– Peptide bond formation occurs when the
polypeptide carried by the peptidyl-tRNA is
transferred to the amino acid carried by the
aminoacyl-tRNA.
– This reaction is catalyzed by constituents of the
large subunit of the ribosome.
8
Ribosomal organisation
• Transfer of the polypeptide generates the ribosome
shown in step 2.
• The deacylated tRNA, lacking any amino acid, lies in
the P site, while a new peptidyl-tRNA has been
created in the A site.
• This peptidyl-tRNA is one amino acid residue longer
than the peptidyl-tRNA that had been in the P site in
step 1.
9
Ribosomal organisation
• Then the ribosome moves one triplet along the
messenger = TRANSLOCATION.
10
Ribosomal organisation
• Then the ribosome moves one triplet along the
messenger = TRANSLOCATION.
• The movement transfers the deacylated tRNA out of
the P site...
11
Ribosomal organisation
• Then the ribosome moves one triplet along the
messenger = TRANSLOCATION.
• The movement transfers the deacylated tRNA out of
the P site...
• moves the peptidyl-tRNA into the P site.
12
Ribosomal organisation
• Then the ribosome moves one triplet along the
messenger = TRANSLOCATION.
• The movement transfers the deacylated tRNA out of
the P site...
• moves the peptidyl-tRNA into the P site.
• Exposes the next codon to be translated in the A
site, ready for a new aminoacyl-tRNA to enter.
13
Ribosomal organisation
• The deacylated tRNA leaves the ribosome via
another tRNA-binding site, the E site (prokaryotes
only).
• This site is transiently occupied by the tRNA en
route between leaving the P site and being released
from the ribosome into the cytosol.
14
The three stages
Protein synthesis divided into the three stages:
1.- Initiation
2.- Elongation
3.- Termination
15
The three stages
Initiation involves the reactions that precede
formation of the peptide bond between the first two
amino acids of the protein.
It requires the ribosome to bind to the mRNA,
forming an initiation complex that contains the first
aminoacyl-tRNA.
This is a relatively slow step in protein synthesis,
and usually determines the rate at which an mRNA
is translated.
16
The three stages
Elongation includes all the reactions from synthesis of
the first peptide bond to addition of the last amino acid.
Amino acids are added to the chain one at a time.
This is the most rapid step in protein synthesis.
17
The three stages
Termination encompasses the steps that are needed to
release the completed polypeptide chain and dissociate
the ribosome from the mRNA.
18
Accessory Factors
• Different sets of accessory factors assist the
ribosome at each stage.
19
Accessory Factors
• Different sets of accessory factors assist the
ribosome at each stage.
• Energy is provided at various stages by the
hydrolysis of GTP.
20
Accessory Factors
• Different sets of accessory factors assist the
ribosome at each stage.
• Energy is provided at various stages by the
hydrolysis of GTP.
• Accessory factors are not covalently linked to
Ribosome (proteins or RNA).
21
Initiation - Prokaryotes
Initiation in bacteria needs 30S subunits and
accessory factors
• Initiation factors (IF in prokaryotes, eIF in
eukaryotes) are proteins that associate with the
small subunit of the ribosome during initiation.
• Initiation of protein synthesis is not a function of
intact ribosomes, but is undertaken by the
separate subunits, which reassociate during the
initiation reaction.
• See “Ribosomal subunit cycle during protein
synthesis in bacteria”.
22
Initiation – Subunit Cycle
Ribosomal subunits cycle during prokaryote protein synthesis.
23
Initiation – Subunit Cycle
Ribosomal subunits cycle during prokaryote protein synthesis.
Bacterial ribosomes engaged in elongating a polypeptide exist as 70S.
24
Initiation – Subunit Cycle
Ribosomal subunits cycle during prokaryote protein synthesis.
Bacterial ribosomes engaged in elongating a polypeptide exist as 70S.
At termination, they are released from the
mRNA to enter a pool of free ribosomes.
25
Initiation – Subunit Cycle
Ribosomal subunits cycle during prokaryote protein synthesis.
Bacterial ribosomes engaged in elongating a polypeptide exist as 70S.
At termination, they are released from the
mRNA to enter a pool of free ribosomes.
In growing bacteria, 80% of ribosomes are
synthesizing proteins only 20% are free.
20%
80%
26
Initiation – Subunit Cycle
Ribosomal subunits cycle during prokaryote protein synthesis.
Bacterial ribosomes engaged in elongating a polypeptide exist as 70S.
At termination, they are released from the
mRNA to enter a pool of free ribosomes.
In growing bacteria, 80% of ribosomes are
synthesizing proteins only 20% are free.
Ribosomes in the free pool can dissociate
into separate subunits.
Free 70S ribosomes are in dynamic
equilibrium with 30S and 50S subunits.
27
Initiation - Prokaryotes
• Initiation occurs at a special sequence on mRNA
called the ribosome-binding site.
• Short sequence of bases that precedes the
coding region.
• The ribosome-binding site is a sequence at
which the small and large subunits associate on
mRNA to form an intact ribosome.
AGGAGG
AUG
28
Initiation - Prokaryotes
The reaction occurs in two steps:

Recognition of mRNA occurs when a small subunit binds to form an initiation
complex at the ribosome-binding site.
29
Initiation - Prokaryotes
The reaction occurs in two steps:


Recognition of mRNA occurs when a small subunit binds to form an initiation
complex at the ribosome-binding site.
Then a large subunit joins the complex to generate a complete ribosome.
30
Initiation Factors - Prokaryotes
• The 30S subunit is involved in
initiation but not by itself
competent to undertake the
reactions of binding mRNA and
tRNA.
• Requires additional proteins
called initiation factors (IF).
• These factors are found only
on 30S subunits, and they are
released when the 30S
subunits associate with 50S
subunits to generate 70S
ribosomes.
31
Initiation Factors - Prokaryotes
• The 30S subunit is involved in
initiation but not by itself
competent to undertake the
reactions of binding mRNA and
tRNA.
• Requires additional proteins
called initiation factors (IF).
• These factors are found only
on 30S subunits, and they are
released when the 30S
subunits associate with 50S
subunits to generate 70S
ribosomes.
NOTE:
•
•
•
•
This behavior distinguishes IF from the
structural proteins of the ribosome.
IF's are concerned solely with
formation of the initiation complex.
They are absent from 70S.
They play no part in the stages of
elongation.
32
Initiation Factors - Prokaryotes
Bacteria use three initiation
factors: IF-1, IF-2 and IF-3.

IF-3 is needed for 30S subunits
to bind to initiation sites in mRNA.
33
Initiation Factors - Prokaryotes
Bacteria use three initiation
factors: IF-1, IF-2 and IF-3.


IF-3 is needed for 30S subunits
to bind to initiation sites in mRNA.
IF-2 binds a special initiator tRNA
and controls its entry into the 30S
P site.
34
Initiation Factors - Prokaryotes
Bacteria use three initiation
factors: IF-1, IF-2 and IF-3.



IF-3 is needed for 30S subunits
to bind to initiation sites in mRNA.
IF-2 binds a special initiator tRNA
and controls its entry into the 30S
P site.
IF-1 binds to 30S and stabilize
the complete initiation complex.
35
Initiation Factors - Prokaryotes
IF-3 has two functions:
1.- ANTI-ASSOCIATION FACTOR.
Stabilizes free 30S subunits.
Which controls the amount of free 30S
subunits, preventing them from
reassociating with 50S subunits.
36
Initiation Factors - Prokaryotes
IF-3 has two functions:
2.- BINDING FACTOR.
Controls the ability of 30S subunits
to bind to mRNA.
Small subunits must have IF-3 in
order to form initiation complexes
with mRNA.
IF-3 must be released from the
30S:mRNA complex in order to
enable the 50S subunit to join.
On its release, IF-3 immediately
recycles by finding another 30S
subunit.
37
Initiation – Initiator tRNA
• A special initiator tRNA starts the
polypeptide chain
• Synthesis of all proteins starts with
the same amino acid: methionine.
• The signal for initiating a
polypeptide chain is a special
initiation codon that marks the
start of the reading frame.
38
Initiation – Initiator tRNA
• Usually the initiation codon is the triplet
AUG, but in bacteria, GUG or UUG are
also used.
• The AUG codon represents methionine
and initiations.
• How are they differentiated by translational
machinery?
39
Initiation – Initiator tRNA
• Two types of tRNA can carry an anticodon for these codons.
• One tRNA is used for initiation, the other for recognizing AUG codons during
elongation.
• Prokaryote initiator tRNA is a special tRNA that is formylated (thus the name
tRNAf).
• It is linked to a Methionine residue (aminoacyl-tRNA), thus the name tRNAfMet
• As Met residue is also formylated it is also abbreviated fMet-tRNAf.
40
Initiation – Initiator tRNA
• Formylation is a two
stage reaction.
• First, tRNA is charged
with Met to generate
Met-tRNAf.
• Then formylation
blocks the free NH2
group.
Formyl blockage prevents incorporation to the A site
(elongation) but can be loaded into P site.
41
Initiation – Initiator tRNA
• This tRNA is used only for initiation.
• It recognizes the codons AUG or GUG (occasionally UUG).
• The codons are not recognized equally well: the extent of initiation declines
50% when AUG is replaced by GUG.
• Inititation declines by 75% when AUG is replaced by UUG.
• The species responsible for recognizing AUG codons in internal locations
is tRNAmMet.
• This tRNA responds only to internal AUG codons.
• Its methionine cannot be formylated.
42
Initiation – Initiator tRNA
• So there are two differences between the initiating and elongating Met-tRNAs: the
tRNA moieties themselves are different; and the amino acids differ in the state
of the amino group.
• The meaning of the AUG and GUG depends on their context.
– When the AUG codon is used for initiation, it is read as formyl-Met; when used
within the coding region, it represents Met.
• The meaning of the GUG codon is even more dependent on its location.
– When present as the first codon, it is read via the initiation reaction as formylMet yet when present within a gene, it is read as Val.
43
Initiation – Initiator tRNA
44
Initiation – Initiator tRNA
• In an initiation complex, the small subunit alone
is bound to mRNA.
• The initiation codon lies within the part of the P
site.
• The only aminoacyl-tRNA that can become part
of the initiation complex is the initiator, which
has the unique property of being able to enter
directly into the partial P site to recognize its
codon.
45
Initiation – Initiator tRNA
• When the large subunit joins the
complex, the initiator fMet-tRNAf
lies in the now-intact P site.
• The A site is available for entry
of the aminoacyl-tRNA
complementary to the second
codon of the gene.
46
Initiation – Initiator tRNA
• Initiation prevails when an AUG (or GUG) codon lies within a ribosome-binding
site, because only the initiator tRNA can enter the partial P site generated
when the 30S subunit binds de novo to the mRNA.
• Only the regular aminoacyl-tRNAs can enter the (complete) A site.
47
Initiation – Initiator tRNA
• What features distinguish the tRNAfMet
initiator and the tRNAmMet elongator?
• Some of these features are needed to
prevent the initiator from being used in
elongation, others are necessary for it to
function in initiation
48
Initiation – Initiator tRNA
Formylation
Both tRNA and amino acid
Formylation is not strictly necessary,
because nonformylated tRNAfMet can
function as an initiator.
Formylation improves the efficiency with
which the tRNAfMet is bound by IF-2.
49
Initiation – Initiator tRNA
Acceptor Stem
base-pairing
The bases C:A on the acceptor stem are NOT
paired in tRNAfMet.
The absence of this pairing makes tRNAfMet
uncapable of being incorporated during
elongation.
Mutations that create a base pair in this position
of tRNAfMet allow it to function in elongation.
It is also needed for the formylation reaction.
50
Initiation – Initiator tRNA
Anti-codon stem
base-pairing
A series of 3 G:C pairs in the stem that
precedes the loop containing the anticodon
is unique to tRNAfMet.
Are required to allow the tRNAfMet to be
inserted directly into the P site.
51
Initiation – Initiator tRNA
The ability of tRNAfMet initiator to
enter the ribosome is controlled by
IF-2.
The 30S subunit carries all the
initiation factors, including IF-2,
which is probably associated with
the P site.
IF-2 specifically forms a complex
with tRNAfMet.
This complex places the tRNA in the
partial P site.
52
Initiation – Initiator tRNA
By forming a complex specifically
with tRNAfMet, IF-2 ensures that only
the initiator tRNA, and none of the
regular aminoacyl-tRNAs,
participates in the initiation reaction.
IF-2 remains part of the 30S subunit
at this stage; it has a further role to
play...
53
Initiation – Initiator tRNA
IF-2 has a ribosome-dependent
GTPase activity.
It sponsors the hydrolysis of GTP in
the presence of ribosomes, releasing
the energy stored in the high-energy
bond.
The GTP is hydrolyzed when the 50S
subunit joins to generate a complete
70S ribosome.
54
Initiation – Initiator tRNA
In bacteria and mitochondria, the formyl residue on the initiator methionine is removed by a
specific deformylase enzyme to generate a normal NH2 terminus.
50% of the proteins the methionine at the terminus is removed by an aminopeptidase,
creating a new terminus from R2 (the second amino acid incorporated into the chain).
When both steps are necessary, they
occur sequentially.
The removal reaction(s) occur rather
rapidly, probably when the nascent
polypeptide chain has reached a
length of 15 amino acids.
55
Initiation – Shine-Dalgarno Sequences
Initiation involves base pairing between mRNA and
rRNA
An mRNA contains many AUG triplets: how is the
initiation codon recognized as providing the starting
point for translation?
When ribonuclease is added to the blocked initiation
complex, all the regions of mRNA outside the
ribosome are degraded.
The protected fragments can be recovered and
characterized.
56
Initiation – Shine-Dalgarno Sequences
The protected initiation sequences of bacteria are ~30 bases long.
Two common features:
The AUG (or less often, GUG or UUG) initiation codon is always included within
the protected sequence.
Within 10 bases upstream of the AUG is a polypurine stretch known as the ShineDalgarno sequence:
5’-AGGAGG-3’
It is complementary to a highly conserved sequence close to the 3’ end of 16S
rRNA:
3’-UCCUCC-5’
57
Initiation – Shine-Dalgarno Sequences
Start Codon AUG
Shine-Dalgarno sequence
(in mRNA):
5’-AGGAGG-3’
Shine-Dalgarno complementary
sequence (16S rRNA):
3’-UCCUCC-5’
58
Initiation – Shine-Dalgarno Sequences
Mutations of any of these sequences impede ribosome binding.
Point mutations in the Shine-Dalgarno sequence prevent mRNA translation.
The sequence at the 3’ end of rRNA is conserved between prokaryotes and
eukaryotes...
59
Initiation – Shine-Dalgarno Sequences
However, eukaryotes have a five-base deletion in the complementary rRNA
sequence.
There is no apparent base pairing between eukaryotic mRNA & 18S rRNA.
This is a significant difference in the mechanism of initiation.
60
Initiation – Shine-Dalgarno Sequences
In bacteria, a 30S subunit binds directly to a
ribosome-binding site.
As a result, the initiation complex forms at a
sequence surrounding the AUG initiation codon.
When the mRNA is polycistronic, each coding
region starts with a ribosome-binding site.
61
Initiation – Shine-Dalgarno Sequences
When ribosomes attach to the first coding region, the subsequent coding regions
have not yet even been transcribed.
By the time the second ribosome site is available, translation is well under way
through the first cistron.
62