Protein Synthesis

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Protein Synthesis Biology 12 Mr. McIntyre
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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
DNA
Transcription
mRNA
5-AUGCCUAGGUACCUAUGA-3
decoded as
5-AUG CCU AGG UAC CUA UGA-3
Translation
Protein
N-MET-PRO-ARG-TYR-LEU-C
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Alanine tRNA
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Generalized tRNA
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Modified Bases Found in tRNAs
UH2
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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 2OH on the
terminal ribose Arg, Cys , Gln, Glu, Ile, Leu,
Met, Trp Tyr, Val Class II - dimeric, acylate
the 3OH 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 the cognate 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 subunit are 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
into mature 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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The large and small subunits come together to
form the ribosome
Mitochondrial or Prokaryotic
Eukaryotic 60S subunit
80S ribosome
40S subunit
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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-acyl tRNA 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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EF-1
Proper reading of the anticodon is the
second important quality control step ensuring
accurate protein synthesis
Elongation factors Introduce a two-step Kinetic
proofreading
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A second elongation factor EF-G or EF-2, drives
the translocation 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.
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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
around the AUG codon, called Shine-Delgarno
sequences, are recognized by an intiation complex
consisting of a Met amino-acyl tRNA, Initiation
Factors (IFs) and the small ribosomal subunit
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GTP hydrolysis by IF2 coincident with release
of the IFs and binding of the large ribosomal
subunit leads to formation of a
complete ribosome,on the mRNA and 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 are 1) Nutrient
availability low nutrient (amino
acids/carbohydrate) downregulates
translation 2) Growth factor signals. stimulat
ion 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
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Control of translation by eIF2 phosphorylation
Stimulated by Amino acid deprivation
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Control of translation by eIF4E availability
The 7MEG 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
Nutrient Limitation
Growth Factors
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Nutritional controls
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Nutritional signals can control both the
recognition of the mRNA and loading of the 40S
subunit.
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Modification of the translation machinery is a
common feature of viral life cycles
e.g. Picornaviruses Polio virus Encephalomyocard
itis 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 the methyl cap binding subunit and
the 40S subunit
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The consequence of this cleavage is that
translation of cellular mRNAs stops
Butthe 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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X 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 used throughout 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