Download Handout 14, 15 - U of L Class Index

tRNA is a link between the mRNA and the polypeptide
being synthesized
During translation proteins are synthesized according to a
genetic message of sequential codons along mRNA
Translation
Transfer RNA (tRNA)
tRNA)– is an interpreter between the two forms of
information – base sequence in mRNA and aminoacid sequence in
polypeptides
tRNA aligns appropriate aminoacids to form a new polypeptide.
To perform this action tRNA must:
•Transfer amino acids from the cytoplasm’s amino acid pool to a
ribosome
•Recognize the correct codons in mRNA
tRNA – an adaptor molecule
Molecules of tRNA are specific for only one particular
amino acid
Each type of tRNA associates a distinct mRNA codon with one of the 20 amino
acids used to make proteins
•One end of tRNA molecule attaches to a specific amino acid
•The other end attaches to an mRNA codon by base-pairing with its anticodon.
Anticodon – a nucleotide triplet in tRNA that base pairs with a complementary
codon in mRNA.
tRNAs decode the genetic message, codon by codon.
Transfer RNA's (tRNA)
Function: carries amino acids to the ribosome for assembly into
polypeptides.
Therefore: translates the mRNA genetic code.
Mechanism
tRNA Structure
•complementary and antiparallel base
pairing of the anticodon on the tRNA
molecule paires with the mRNA codon.
•determines which amino acid is added by
the ribosome to the growing polypeptide.
Structure
•Single strand of RNA, 80 bp long
•Folded into clover leaf configuration,
driven by complementary base pairing.
•Anticodon loop
•Amino acid-binding site (3'-end)
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All tRNAs have a similar structure
The shortest functional RNAs know: the smallest are 74 nt in
length, the largest – ralely more than 90 nt.
They were among the first RNAs discovered
Clover leaf:
Acceptor arm, formed by 7bp between the 5’and 3’ ends of the
molecule
The aa attaches at the extreme 3’ end of the tRNA, to the
adenosine of the invariant of terminal CCA sequence
The D arm – named after modified nucleoside dihydrouridine,
which is always present in this structure
The anticodon arm, containing triplet called anticodon that will
base-pair with mRNA during translation
The V-loop, which contains 3-5 nts or 13-21 nts.
The TΨC arm, named after the sequence of thymidinepseudouridine-cytosine, which it always contains
An aminoacyl-tRNA synthetase joins a
specific amino acid to a tRNA.
Linkage of the tRNA and amino acid is an
endergonic process that occurs at the expense
of ATP.
AminoacylAminoacyl-tRNA synthetase – enzyme that catalyzes the
attachment of an amino acid to its tRNA
The correct linkage between tRNA and its designated amino acid must
occur before the anticodon pairs with its complementary mRNA
codon.
Each of the 20 amino acids has a specific aminoacyl-tRNA synthetase
The proper synthetase attaches an amino acid in two steps. ATP is
needed.
1. Activation of the amino acid with AMP. The synthetase’s active
site binds the amino acid and ATP; the ATP loses two phosphate
groups and attaches to the amino acid as AMP.
2. Attachment of the amino acid to RNA. The appropriate tRNA
covalently bonds to the amino acid, displacing AMP from the
enzyme’s active site.
The aminoacyl-tRNA complex releases from the enzyme and
transfers its amino acid to a growing polypeptide on the ribosome.
AminoacylAminoacyl-tRNA synthetase activity
1. The active site of the enzyme binds the
amino acid and an ATP molecule.
2. The ATP loses two phosphate groups and
joins to the amino acid as AMP (adenosine
monophosphate).
3. The appropriate tRNA covalently bonds to
the amino acid, displacing the AMP from the
enzyme's active site.
4. The enzyme releases the aminoacyl tRNA,
also called an "activated amino acid."
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AminoacylAminoacyl-tRNA synthetase has affinity to its tRNA
This is due to the extensive interaction between the two, covering
25nm2 of the surface area and involving the acceptor arm and the
anticodon loop of the tRNA, as well as individual nucleotides in
the D and the TΨC arm
The interaction between enzyme and aa is less extensive, the aa is
smaller, several pairs of aa are structurally similar.
Errors do occur, but at a low rate.
Genetic code – the set of three-base code words (codons) in
mRNA that stand for the 20 aa in proteins.
•Code is non-overlapping - each base is a part of only one
codon
•Code is devoid of gaps, or commas -each base is a part of
codon.
Frameshift mutations
When the enzyme attaches the wrong aa to a tRNA, this aa is
subsequently transformed to correct by a separate reaction.
This was first discovered in B. megaterium.
Glutamic acid – glutamine conversion by transamidation
reaction.
Asparagine-tRNA from aspartic acid-tRNA in other bacteria (not
E. coli)
Breaking the genetic code
It was broken by using synthetic messengers of synthetic
trinucleotides and observing the polypeptides synthesized or aminoacyl-tRNAs bound to ribosomes.
Code is degenerate:
•There are 64 codons in all.
•Three are stop-codons
•The rest code for amino acids.
The genetic code
There are only about 45 distinct types of tRNA.
This is enough to translate 64 codons, as some tRNA recognize
two or three codons specifying the same amino acid.
This is possible because the rules are relaxed between the third
base of an mRNA codon and the corresponding base of a tRNA
anticodon.
This exception to the base-pairing rule is called wobble.
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Ribosomes
Ribosomes coordinate the pairing of tRNA anticodons to mRNA
codons
Ribosomes have two subunits – large and small – separated when
not involved in protein synthesis.
Ribosomes are composed of about 60% ribosomal RNA (rRNA)
and 40% protein.
Ribosomes
In addition to an mRNA binding site, each ribosome has three
tRNA binding sites (P, A and E).
•The P site holds the tRNA carrying the growing polypeptide
chain
•The A site holds the tRNA carrying the next amino acid to be
added
•Discharged tRNAs exit the ribosome from the E site.
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Ribosomes
Large and small subunits of the ribosomes are
•Constructed in the nucleolus
•Dispatched through nuclear pores to cytoplasm
•Once in the cytoplasm, are assembled into functional ribosomes
only when attached to an mRNA
Compared to eukaryotic ribosomes, prokaryotic ribosomes are
smaller and have a different molecular composition.
The anatomy of a ribosome.
(a) A functional ribosome consists
of two subunits, each an aggregate
of ribosomal RNA and many
proteins.
(b) A ribosome has an mRNAbinding site and three tRNAbinding sites, known as the P, A,
and E sites.
(c) A tRNA fits into a binding site
when its anticodon base-pairs with
an mRNA codon. The P site holds
the tRNA attached to the growing
polypeptide. The A site holds the
tRNA carrying the next amino acid
to be added to the polypeptide
chain. Discharged tRNA leaves via
the E site.
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Ribosomes
Ribosome structure
Microscopy led to initial progress in understanding structure of
ribosomes.
In 1940s – first photo-micrographs of bacterial ribosomes - oval –
structures, 29 x 21 nm
Eukaryotic ones – bigger, about 32 x 22 nm
Ultra centrifugation was used to measure sizes of ribosomes and
their composition.
Each ribosome has two subunits:
In eukaryotes – 60S and 40S
In bacteria – 50S and 30S
NB: sedimentation coefficients are not additive because they
depend upon shape as well as upon mass: it is perfectly acceptable
for the intact ribosomes to have the S coefficient less than a sum of
subunits.
Sedimentation coefficients of intact eukaryotic ribosomes – 80S
Prokaryotic – 70S.
They can be broken into smaller components.
Ribosomes
The large subunit contains three rRNAs in eukaryotes: 28S, 5.8S and
5S rRNA
Steps of translation
In bacteria – only rRNAs two 23S and 5S rRNA. In bacteria the
equivalent of the eukaryotic 5.8S rRNA is contained within the 23S
rRNA.
Building of a polypeptide, or translation occurs in
three stages:
The small subunit contains a single rRNA in both types of organisms:
an 18S rRNA in eukaryotes and a 16S rRNA in bacteria.
1. Initiation
Both subunits are associated with a variety of ribosomal proteins:
Eukaryotes
Bacteria
60S – 50 proteins
50S – 34 proteins
40S – 33 proteins
30S – 21 proteins
2. Elongation
3. Termination
The proteins of the small subunits are called S1, S2 etc.; those of the
large one – L1, L2, etc. There is one of the proteins per each ribosome,
except for L7 and L12, which are present as dimers.
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Initiation
Initiation brings together mRNA, a tRNA attached to the first
amino acid (aa) (initiator tRNA, the 1st aa is always
methionine), and the two ribosomal subunits.
Initiation in bacteria
The main difference between initiation of translation in bacteria and
eukaryotes is that in bacteria the translation initiation complex is
built up directly over the initiation codon, the point at which protein
synthesis will begin.
Eukaryotes, use a more indirect process for locating the initiation
point.
In bacteria, the process initiates when a small subunit, in conjunction
with the translation initiation factor IF-3, attaches to the ribosome
binding site –Shine-Dalgarno sequence.
Shine-Dalgarno sequence – a short target site, consensus sequence
5’-AGGAGGU-3’ in E. coli, located about 3-10 nucleotides
upstream the initiation codon, where the translation begins.
Initiation in bacteria
The ribosome binding site is complementary to the region at the
3’-end of the 16S rRNA, the one present in the small subunit.
This base pairing is involved in the attachment of the small
subunit to the mRNA.
Attachment positions the small subunit over the initiation codon.
This codon is usually 5’-AUG’3’, but sometimes may be 5’GUG-3’, or 5’-UUG-3’.
All three codons are recognized by the same initiator tRNA, the
last two by wobble.
Initiation of translation in prokaryotes
•The modification attaches a formyl group, -COH, to the amino-group
which means that only the carboxyl group of the initiator methionine is
free to participate in peptide bond formation.
•This ensures that polypeptide bond synthesis can take place only in the
N→C direction.
•The initiator tRNA Met is brought to the small subunit of the ribosome
by a second initiation factor, IF-2, along with the molecule of GTP, the
latter acts as energy source.
•Only tRNA Met is only able to decode initiation codon.
•NB: During elongation the internal AUG codons are recognized by
different tRNA Met, carrying unmodified methionine.
•Completion of initiation phase: when IF-1 binds to and stabilizes
initiation complex, enabling the large subunit to attach.
•Attachment of the large subunit requires energy.
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Summary of prokaryotic
translation initiation
1. Dissociation of 70S into 50S
and 30S subunits, under the
influence of IF-1.
2. Binding of IF-3 to the 30S
subunit, which permits reassociation between the
ribosomal subunits.
3. Binding of IF-1, IF-2 and
GTP alongside IF-3.
Summary of prokaryotic
translation initiation
4. Binding of mRNA and tRNAi
Met to form the 30S initiation
complex. These components
can bind in either order, but
IF-2 sponsors tRNAi Met
binding, and IF-3 sponsors
mRNA binding. In each case
the other factors also help.
5. Binding of 50S subunit, with
the loss of IF-a and IF-3.
6. Dissociation of IF-2 from the
complex, with simultaneous
hydrolysis of GTP. The
product is the 70S initiation
complex, ready to begin
elongation.
Initiation in eukaryotes is mediated by the cap structure and
poly(A)tail
Simplified version of the scanning model for translation initiation
The 40S ribosomal subunit, alongside with factors, tRNAi Met and GTP
recognize the m7G cap at the 5’-end of an mRNA and allow the
ribosomal subunit to bind at the end of the mRNA.
The 40S subunit is scanning the mRNA toward the 3’-end, searching
for the initiation codon, melting the stem loop structure in its way.,
The ribosomal subunit locates an AUG initiation codon and stops
scanning. Now the 60S ribosomal subunit can join the complex and
initiation can occur.
Initiation in eukaryotes
Only small number of eukaryotic mRNAs have internal ribosome
binding sites.
The small subunit of the ribosome makes its initial attachment at
the 5’-end of the mRNA and scans along the RNA sequence to find
the AUG.
The first step involves the pre-initiation complex:
40S subunit of the ribosome, GTP, tRNAi Met and eukaryotic
factor eIF-2 (a trimer of three different proteins).
After assembly the pre-initiation complex associates with the 5’end
of mRNA.
This step requires the cap binding complex.
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Initiation in eukaryotes
Initiation in eukaryotes
After attachment to 5’end the complex is called initiation complex.
The attachment is also influenced by polyA. This is thought to be
mediated by polyadenylate-binding protein (PADP) which is
attached to polyA.
It can scan the RNA.
PADP forms association with eIF-4G, requiring the mRNA bends
back on itself.
eIF-4A and eIF-4B: eIF-4A and possible eIF-4B have helicase
activity and are able to break intramolecular base-pairs in mRNA
freeing the passage for ribosome.
With artificially uncapped DNA the PADP interaction is sufficient
to load pre-initiation complex.
The initiation codon, the 5’-AUG-3’ is within the consensus
sequence – 5’-ACCAUGG-3’, called Kozak sequence.
Leader sequences in eukaryotes are long – up to 100 or more bp,
have structures – hairpins and other.
When initiation complex is positioned over the initiation codon, the
large subunit attaches.
The elongation cycle of translation - overview
Elongation is similar in eukaryotes and
prokaryotes
Several proteins called elongation factors take part in this threestep cycle which adds amino acids one by one to the initial
amino acid:
1. Codon recognition.
2. Peptide bond formation.
3. Translocation.
Not shown are the elongation factors and GTP.
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Elongation
1. Codon recognition.
•
The mRNA codon in the A site of the ribosome forms hydrogen
bonds with the anticodon of an entering tRNA carrying the next
amino acid in the chain.
•
An elongation factor EF-Tu directs tRNA into the A site in
bacteria. In eukaryotes – eEF-1 (4 subunits: eEF-1α, eEF-1β,
eEF-1γ, eEF-1δ)
•
eEF-1α consists of eEF-1α1 and eEF-1α2
•
Hydrolysis of GTP provides energy for this step.
2. Peptide bond formation.
•A peptide bond is formed between the polypeptide in the P site
and the new amino acid in the A site by a peptidyl transferase.
This reaction requires hydrolysis of GTP bound to EF-Tu, or
eEF-1.
•This inactivates EF-Tu, it is ejected from the ribosome and
regenerated by EF-Ts. No eukaryotic homology of EF-Ts is
known, but possibly one of the subunits of the eEF-1 has such
activity.
•Peptidyl transferase activity appears to be one of the rRNAs in
the large ribosomal subunit .
•The polypeptide separates from its tRNA and is transferred to
the new amino acid carried by the tRNA in the A site.
Elongation
Elongation of translation
1.
3. Translocation.
•The tRNA in the A site, which is now attached to the growing
peptide, is translocated to the P site. Simultaneously, the tRNA
that was in the P site is translocated to the E site and from there it
exits the ribosome.
•During this process, the codon and anticodon remain bonded, so
that mRNA and the tRNA move as a unit, bringing the next codon
to be translated into the A site.
•The mRNA is moved through the ribosome only in the 5’ to 3’
direction.
•Translocation requires GTP hydrolysis and is mediated by EF-G
in bacteria and by eEF-2 in eukaryotes.
2.
EF-Tu with
GTP binds
amino-acyl
tRNA to the A
site.
Peptidyl
transferase
forms bond
between
peptide in the
P site and the
newly arrived
amino-acyl
tRNA in the Asite. This
lengthens
peptide and
shifts it to the
A site.
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Elongation of translation
3.
EF-G, with
GTP,
translocates
the growing
peptidyl
tRNA, with
its mRNA
codon to the
P site.
Termination of translation
3. Eukaryotes have just one factor - eRF.
4. In bacteria – process is energy-independent.
5. In eukaryotes – requires hydrolysis of GTP.
6. Termination results in release of completed polypeptide from
tRNA in the P site, and dissociation of the translation complex.
7. Ribosome subunits enter the cytoplasmic pool where they remain
until used again in another round of translation.
Termination of translation
1. A site is entered not by tRNA but by a protein called release
factor. The release factor hydrolyzes the bond between the tRNA
in the P site and the last amino acid of the polypeptide chain. The
polypeptide is thus freed from the ribosome.
2. Bacteria have 3 of release factors:
RF-1 which recognizes the termination codons UAA and UAG
RF-2 which recognizes UAA and UGA
RF-3 which plays supporting role.
Post-translational processing of proteins
Translation is not the end of the gene expression. The polypeptide
that emerges is inactive, but before it must undergo at least the first of
the following steps:
Protein folding. The polypeptide is inactive until it is folded into its
correct tertiary structure. In cells folding is aide by molecular
chaperones. In E. coli, the chaperones are divided into 2 groups:
The Hsp70 chaperones, which include proteins called Hsp70,coded
by dnaK gene, and Hsp40 coded by dnaJ gene and the GrpE.
The chaperons bind to hydrophobic regions of the proteins, including
proteins that still are being translated. They prevent aggregation by
holding the protein in an open confirmation until it is completely
synthesized and ready to fold. They also are involved in other
processes that require shielding of hydrophobic regions.
The chaperonins, the main version of which is E. coli is
GroEL/GroEs complex. The complex is a multisubunit structure that
looks like a hollowed-out bullet. Protein enters unfolded and exists
folded. Mechanism - unknown
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Post-translational processing of proteins
Eukaryotic folding
Proteins equivalent to chaperons and chaperonins have been
identified.
Eukaryotic folding makes less use of chaperonins, and more
depends upon the action of Hsp70 chaperons.
Post-translational processing of proteins
Proteolytic cleavage. Some proteins are processed by cutting
events carried out by enzymes called proteases. These cutting
events may remove segments from one or both ends of the
polypeptide, resulting in a shortened form of a protein, or they may
ct polypeptide into a number of different segments, each one of
which is active
Chemical modification. Individual amino acids in polypeptide
might be modified by attachment of new chemical groups.
Intein splicing. Inteins are intervening sequences in some proteins,
similar in a way to introns in mRNAs. They have to be removed
and the exteins ligated in order for the protein to become active.
Reading:
Chapters 17, 18.
References:
R. Weaver, Molecular Biology, 2005.
T. A. Brown, Genomes, 1999.
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