Download mRNA translation

Tutorial II
Program
Gene expression:
mRNA translation and protein
synthesis
Control of gene transcription and RNA
processing
Piergiorgio Percipalle, PhD
mRNA translation and protein synthesis
KAROLINSKA INSTITUTET
Department of Cell and Molecular Biology
Medical Nobel Institute
Highlights
mRNA translation
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What is mRNA translation?
Molecular mechanisms
Components
Medical relevance
Regulation
mRNA functions as the intermediate
between DNA and protein
How does the information stored
in a linear sequence of nucleotides
in the mRNA translate to a linear
sequence of amino acids to obtain
proteins?
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Transcription
Same language:
1 dNTP ≡ 1 rNTP
Protein synthesis
Different languages:
1 rNTP ≠ 1 aa
Specific problems
All cells must be able to translate the nucleotidebased language into proteins
The translation can not be done letter by letter
because there are 4 bases in the DNA/RNA and 20
different amino acids
RNA translation
The genetic code
The genetic code
mRNA is read in codons (nucleotide triplets), t ex
ATG
Every codon specifies an amino acid or a ”STOP” in
the translation process
The genetic code is universal
The genetic code is redundant since many amino
acids are specified by several codons.
Black letters: codons (nucleotide triplets)
Red letters: amino acids
Adaptor molecules match amino acids
to mRNA codons
The adaptor molecule is called transfer-RNA (tRNA)
How are nucleotides coupled
with amino acids?
2
The 3D structure of tRNA is L-shaped
and exhibits binding sites for both
amino acids and codons
The genetic code
3’ end
Amino acid
binding site
Antico
don
Codon binding
site
The ”Wobble”
Wobble” effect in base pairing
between codons and anticodons
Black letters: codons (nucleotide triplets)
Red letters: amino acids
A specific enzyme loads
tRNA molecules with an amino acid
The enzyme is called amino acyl t-RNA synthetase
1)
2)
The genetic code is translated
with the help of two different adaptors
The new amino acids are incorporated
in the growing polypeptide chain
Amino acyl tRNA
Net result: one amino acid is chosen by one codon
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Ribosomes
A
Where does translation take place?
B
C
Eukaryotic
ribosome
Ribosomes
Ribosomes are very complex catalytic machineries
containing more than 50 different proteins as well as
ribosomal RNA (rRNA)
Active ribosomes conist of a large and a small
subunit
Ribosomes are very conserved between prokaryotic
and eukaryotic organisms
In eukaryotes, active ribosomes are only present in
the cytoplasm
Prokaryotic
ribosome
Antibiotics and mRNA translation
Antibiotics are designed against processes and
structure which are unique for microbes and are not
found in eukaryotes
There are slight differences between eukaryotic and
prokaryotic ribosomes and these differences are
targeted by antibiotics
There are antibiotics (eg. kanamycin)
kanamycin) which
specifically block bacterial ribosomes
Protein synthesis is a dynamic
process
How can the ribosome facilitate
and coordinate all the molecular
mechanisms that take place for
efficient mRNA translation?
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There are three main steps
in mRNA translation
Ribosomes contain four binding sites
Binding of amino acyl-tRNA to A-site
tRNA binding sites
The C-terminus end of the peptide in the
P-site is coupled to the N-terminus of
amino acid in the A-site
mRNA binding site
Conformational changes cause the shift
of the mRNA by exactly three nucleotides
so that a new AA-site becomes available
Translation of an mRNA molecule…..
….require the help of elongation factors
A
B
D
E
Peptidyl transferase
C
F
Conformational changes
in the mRNA
Initiation and termination of translation
requires specific signals
Initiation requires a specific AUG codon, which
exclusively binds to a tRNA carrying a methionine
Translation is terminated when stop codons (UAA,
UAG, UGA) are encountered by the ribosome
Initiation requires initiation factors
Initiator tRNA binds the small ribosomal
subunit
The small ribosomal subunit recognizes
the 5’-cap structure in the mRNA (it also
needs initiation factors eIF4E and
eIF4G)
The small ribosomal subunit scans the
mRNA until it reaches the AUG codon
None of the tRNA molecules recognizes stop codons
The large ribosomal subunit binds the small
ribosomal subunit to reconstitute an active
ribosome. The initiation factors are
dissociated
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Initiation of translation
Termination of translation
A
D
B
E
The proteins which are
ready leave ribosomes
C
F
The ribosomal subunits dissociate
Proteins are made in polyribosomes
The synthesis of most proteins takes between 20
seconds and few minutes
A
Quality control of newly
synthesized proteins ?
B
Proteins are folded co-translationally
Proteins acquire their secondary structure during the
translation process
Several mechanisms garantee
correct protein folding
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Abnormally folded proteins can aggregate and give
rise to disease
A long way from DNA to protein
Prion, Huntington and Alzheimer are the result of
accumulation of wrongly folded proteins which build
filaments that can not be degraded and impair cellular
function
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