lects. 16&17 translation
TRANSCRIPT
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Translation: From messenger RNA to protein:
The information encoded in the DNA is transferred to
messenger RNA and then decoded by the ribosome to
produce proteins.
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5¶-ATGCCTAGGTACCTATGA-3¶
3¶-TACGGATCCATGGATACT-5¶
5¶-AUGCCUAGGUACCUAUGA-3¶
5¶-AUG CCU AGG UAC CUA UGA-3¶
N-MET-PRO-ARG-TYR-LEU-C
DNA
Transcription
decoded as
Translation
mRNA
Protein
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Alanine tRNA
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Generalized tRNA
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=UH2
Modified Bases
Found in tRNAs
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tRNAs are activated by amino-acyl tRNA synthetases
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Structure of an amino acyl-tRNA synthetase bound to a tRNA
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One mechanism for maintaining high fidelity of protein
synthesis is the high fidelity of aa-tRNA synthetases
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Amino-acyl tRNA synthetases:
One synthetase for each amino acid
a single synthetase may recognize multiple tRNAs
for the same amino acid
Two classes of synthetase.Different 3-dimensional structures
Differ in which side of the tRNA they recognize
and how they bind ATP
Class I - monomeric, acylates the 2¶OH on the terminal ribose
Arg, Cys , Gln, Glu, Ile, Leu, Met, Trp Tyr, Val
Class II - dimeric, acylate the 3¶OH on the terminal ribose
Ala, Asn, Asp, Gly, His, Lys, Phe, Ser, Pro, Thr
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Two levels of control to ensure that the proper amino acid
is incorporated into protein: 1) Charging of the proper tRNA
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2) Matching thecognate tRNA to the
messenger RNA
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Incorporation of amino acids into polypeptide chains I
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Incorporation of amino acids into polypeptide chains II
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Protein synthesis occurs on ribosomes
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Protein synthesis occurs on ribosomes
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and mitochondria
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Ribosome Assembly
The proteins of each
ribosomal subunitare organized around
rRNA molecules
16S rRNA
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Ribosome Assembly: takes place largely in a specialized domain of
the nucleus, the nucleolus
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In the nucleolus, RNA polymerase I transcribes the rDNA repeats
to produce a 45S RNA precursor
The 45S precursor
is processed
and cleaved intomature rRNAs and
ribosomal proteins
then bind to generate
the large and small
ribosomal subunits
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23S rRNA secondary structure
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3D organization of the eukaryotic large subunit rRNA
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Ribosomal Proteins decorate the surface of the ribosome
Large subunit. Grey = rRNA Gold = ribosomal proteins
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Ribosomal proteins often have extensions that snake into
the core of the rRNA structure
Crystal structure of L19 L15 (yellow) positioned in a fragment
of the rRNA (red)
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The ribosomal proteins are important for maintaining the
stability and integrity of the ribosome, but NOT for catalysis
ie. the ribosomal RNA acts as a ribozyme
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Mitochondrial
or ProkaryoticEukaryotic 60S subunit 80S ribosome 40S subunit
The large and small subunits come together to form the ribosome
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The association of the large and small subunits creates the
structural features on the ribosome that are essential for
protein synthesis
Three tRNA binding
sites:
A site=
amino-acyltRNA binding site
P site = peptidyl-tRNA
binding site
E site = exit site
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In addition to the APE sites there is an mRNA binding groove
that holds onto the message being translated
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There is a tunnel through the large subunit that allows the
growing polypeptide chain to pass out of the ribosome
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Peptide bond formation is catalyzed by the large subunit rRNA
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Peptide bond formation is catalyzed by the large subunit rRNA
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Incorporation of the correct amino acyl-tRNA is determined
by base-pairing interactions between the anticodon of the
tRNA and the messenger RNA
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Proper reading of the
anticodon is the second
important quality control
step ensuring accurate
protein synthesis
=EF-1
Elongation factors
Introduce a two-step
³Kinetic proofreading´
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A second elongation factor
EF-G
or EF-2, drives thetranslocation of the ribosome
along the mRNA
Together GTP hydrolysis
by EF-1 and EF-2 help drive
protein synthesis forward
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Termination of translation
is triggered by stop codons
Release factor enters
the A site and triggers
hydrolysis the peptidyl-tRNA
bond leading to release of the protein.
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Release of the protein causes
the disassociation of the
ribosome into its constituent
subunits.
R l F i l l i i f RNA
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Release Factor is a molecular mimic of a tRNA
eRF1 tRNA
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Initiation of Translation
Initiation is controlled differently in prokaryotic and
eukaryotic ribosomes
In prokaryotes a single transcript can give rise to multiple proteins
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In prokaryotes, specific
sequences in the mRNA aroundthe AUG codon, called
Shine-Delgarno sequences,
are recognized by an intiation
complex consisting of a Met
amino-acyl tRNA, InitiationFactors (IFs) and the small
ribosomal subunit
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GTP hydrolysis by
IF2 coincident withrelease of the IFs and
binding of the large
ribosomal subunit leads
to formation of a complete
ribosome,on the mRNAand ready to translate.
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Eukaryotic mRNAs have a distinct structure at the 5¶ end
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Structure of the 7-methyl guanosine cap
The 7me-G cap is required
for an mRNA to be
translated
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In contrast, Eukaryotes
use a scanning mechanism
to intiate translation.
Recognition of the AUG
triggers GTP hydrolysis by eIF-2
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GTP hydrolysis by
eIF2 is a signal for
binding of the large
subunit and beginning
of translation
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Messenger RNAs are translated on polyribosomes
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Protein synthesis is often regulated at the
level of translation initiation
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An example of control of specific mRNAs: regulation by iron (Fe):
Ferritin is a cytosolic iron binding protein expressed when
iron is abundant in the cell.
Transferrin receptor is a plasma membrane receptor important
for the import of iron into the cytosol.
They are coordinately regulated, in opposite directions, by
control of protein synthesis.
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Regulation by iron (Fe):
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There is also general control of translational initiation.
ie. all transcripts of the cell are effected (though the relative
effect differs between specific mRNAs)
Global downregulation or upregulation can occur in response
to various stimuli the most common are1) Nutrient availability
low nutrient (amino acids/carbohydrate)
downregulates translation
2) Growth factor signals.
stimulation of cell division upregulates translation
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General control of translational initiation is exerted through
two primary mechanisms.
Control of the phosphorylation of eIF2
Control of the phosphorylation of eIF4 binding proteins
Control of translation b eIF2 phosphor lation
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Control of translation by eIF2 phosphorylation
Stimulated by
Amino acid deprivation
Control of translation by eIF4E availability
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Control of translation by eIF4E availability
The7ME
G cap binding subunit of eIF4, eIF4E, is sequestered by eIF4E binding protiens (4E-BPs). The binding of these
proteins is regulated by their phosphorylation state
GrowthFactors
NutrientLimitation
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Nutritional signals can control both the recognition of the mRNA
and loading of the 40S subunit.
Nutritional
controls2
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Modification of the translation machinery is a common
feature of viral life cycles
e.g. Picornaviruses
Polio virusEncephalomyocarditis virus
Picornaviruses have single stranded RNA genomes.
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Poliovirus Life Cycle
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The poliovirus genome is translated into a single,
large polyprotein that then auto-proteolyzes itself into
smaller proteins.
One of these proteins, viral protease 2A cleaves
the translation initiation factor eIF4G so that it
can no longer function as a bridge between themethyl cap binding subunit and the 40S subunit
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The consequence of this cleavage is that translation of
cellular mRNAs stops
But«the viral RNA is still translated due to the presence of
an internal ribosomal entry site (IRES). This acts like a
bacterial initiation site to allow Cap-independent initiation
from internal AUG codons.
What is X?
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³X´ is not a protein, as suggested
by the textbook model at right,rather it is a structure in the mRNA
itself that can bind to the remaining
fragment of eIF4G
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Some cellular mRNAs are also translated using IRESs
During G2/M phase of the cell cycle, translation is generally
downregulated by activation of 4E-BPs. Many proteins expressed
during this period bypass this control by using IRES elements
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Ribosomal Frameshifting
Because translation
uses a triplet code,there are three potential
reading frames in
each mRNA
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As the ribosome translocates, it moves in three nucleotide
steps, ensuring that the frame defined by the AUG
is usedthroughout translation
If the ribosome moves 1 or 2 (or 4 or 5) nucleotides
this produces a frameshift
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Many retroviruses induce ribosomal frameshifting in
the synthesis of viral proteins
e.g. HIV
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Translation Inhibitors are important antibiotics