Biochemistry
Lecture 14 2008 (J.S.)
Posttranslational modifications of proteins
Glycoprotein synthesis
RNA translation - proteosynthesis
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The genetic code
Codons in mRNA (read in the direction 5´→ 3´):
– Triplets of bases,
– unambiguous code,
– degeneracy of the code,
– non-overlapping,
– in a continuous sequence.
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Degeneracy of the genetic code
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The reading frames
Only one of the three possible frames is the right one,
it begins at the start codon AUG recognized by the Met-tRNAMet
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Consequences of mutations
Mutations are structural alterations in the genome that can lead to an
alteration in the sequence of a protein translated from it, if they are
transcribed into mRNA.
Types of mutations:
– point mutations, alterations in a single base, which
- can result in change of the sense (a single amino acid replacement,
e.g. GCA→CCA results in Arg→Pro),
- can be neutral, causing no change (e.g. CGA→CGG, both encode Arg),
- can terminate translation, if result in stop-codons (nonsense).
– insertions of one or more nucleotides, and
– deletions of one or more nucleotides.
The result depends on the number of inserted or deleted nucleotides.
If three nucleotides (or more triplets) are inserted/deleted without a change of the
reading frame, polypeptides with inserted/deleted amino acyl residues will be
synthesized.
If one or two nucleotides are inserted/deleted, the result is a "frame-shift mutation"
that gives nonsense codons, distinct primary structure of proteins, etc.
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tRNA molecules act as adaptors in translation,
amino acids cannot react directly with bases of nucleotides.
Helix stacking in L-shaped tRNA structure
General structure of tRNA molecules
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Schematic representation of the arrangement of
ribosomal RNA and ribosomal protein within the
ribosome.
Ribosomes
30 S
50 S
70 S
Ribosome of the E. coli
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Eukaryotic ribosome
80 S
40 S
60 S
18 S rRNA
~ 33 proteins
28 S, 5.8 S, 5 S rRNA
~ 49 proteins
SMALL subunit LARGE subunit
Sedimentation constant 40 S 60 S
rRNA types 18 S (1874 bases) 28 S (4718 bases)
5.8 S (160 bases)
5 S (120 bases)
Proteins S1 – S33 L1 – L49
RNA mass fraction 0.50 0.65
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Three binding sites for tRNA
E exit site
P peptide site
A amino acid site
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Activation of amino acids
Synthesis of aminoacyl-tRNAs ("charging" of tRNAs)
Aminoacyl-tRNA synthetases (at least 20 distinct enzymes in cells)
exhibit the very high degree of specifity for amino acids.
The enzyme molecule recognizes both a specific amino acid and a specific tRNA.
These enzymes discriminate accurately, the overall rate of occurence of errors in
translating mRNA is less than 1 in 10 000.
This high specifity is oft called the 2nd
genetic code.
It depends on specific location of some bases in tRNA molecules,
not on the sole anticodon.
-3‘- OH
aminoacyl-tRNA synthetase tRNAAA
aminoacyl-tRNAAA
amino acid
-3‘-
Enzyme–Ado-P–C–CH
O R
NH2
(aminoacyl adenylate)
R–CH
NH2
COOH
ATP PPi
Enzyme
AMP
Enzyme
R
NH2
–C–CH
O
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The initiation of protein synthesis
Formation of the 80 S initiation complex eIFs – eukaryotic initiation factors
AUG
5´-m7
Gppp cap of mRNA binds CBP (cap-binding protein)
and several eIFs; mRNA binds to preinitiation complex which scans
mRNA till reaching the start codon AUG.
eIF2
GTP
Charged Met-tRNAMet
binds eIF2 and GTP
Preinitiation complex
Small ribosomal subunit with eIF1A and eIF3
is attached to Met-tRNA
GTP
AUG
GTP
40S initiation complex
is formed in this way:
GTP is hydrolyzed, GDP, eIF2, eIFs released
and 60S subunit attached
giving so 80S initiation complex:.
GDP
80S initiation complex
AUG GUC
eIF2
eIFs
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The elongation cycle
80S initiation complex
AUG GUC
EF1α-GTP
AUG GUC
tRNAMet
released
Peptidyl transferase
activity of the ribosome
AUG GUC
Translocation
of 60S subunit
Translocation
of 40S subunit
EF1α-GDP + Pi
EF2 + GTP
EF2 + GDP
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The termination of protein synthesis
Elongation continues as far as the binding site A reaches a stop codon.
Releasing factors (eRFs) bind to the site A and peptidyl transferase catalyzes
the hydrolysis of the ester bond between the polypeptide and tRNA.
Ribosomal subunits dissociate, mRNA, "uncharged" tRNA, eRFs and GDP
are released.
UAA
UAA
UAA
eRFs
GTP
+ 2H2O
GDP + Pi
eRFs
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Origin of aminoacyl-tRNA ATP → AMP + 2 Pi 2
Binding of aminoacyl-tRNA to site A GTP → GDP + Pi 1
Translocation of ribosome GTP → GDP + Pi 1
ATP equivalent
Formation of one peptide bond 4 ATP
Energy required in protein synthesis
Velocity of ribosomal protein synthesis
In prokaryotes, approximately hundreds of peptide bonds are formed per second.
In eukaryotes, approximately 1 – 2 peptide bonds per second.
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Folding of proteins
A nascent polypeptide chain grows out from the larger ribosomal subunit (about
40 AA residues are hidden in the subunit) following a leader sequence – a signal
peptide and the folding of the native three-dimensional conformation may begin.
The details of how proteins make the transitions from unfolded structures to
unique conformations in the native forms have not been fully elucidated.
For even a small protein, it would take much too long to fold properly by randomly trying
out all possible conformations.
Proteins follow a partly defined folding pathway consisting of intermediates
between the fully unfolded protein (polypeptide chain) and its native conformation.
There is the tendency to retain partly correct intermediates,
which can interact with one other, leading to increased stabilization
(the nucleation-condensation model).
Folding of proteins is supported by molecular chaperones that stabilize unfolded
or partially folded intermediates, allowing them time to fold properly, and prevent so
faulty folding and inappropriate interactions (formation of non-functional
structures).
Many of chaperons are so-called "heat shock proteins" (inducible by elevated
temperature and various chemicals – conditions that cause unfolding of newly synthesized
proteins).
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Free ribosomes synthesize proteins that remain within the cell, either
within the cytoplasm, or directed to organelles bounded by a double membrane to
the nucleus and mitochondria.
The synthesis of all proteins begins on free ribosomes in the cytoplasm.
Protein synthesis continues on free ribosomes in the cytoplasm till an entire
signal sequence of the nascent chain that directs the ribosome to the
endoplasmic reticulum appears in the cytoplasm.
Ribosomes bound to the ER usually synthesize proteins destined to leave the cell
(secretory proteins), lysosomal proteins and proteins of the plasma membrane.
Sorting of proteins
A fundamental component of the sorting system are the signal sequences
(not always specific) at the N-end of the growing polypeptide chains.
These targeting sequences direct proteins (preproteins) to their destinations and
are subsequently removed.
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SRP receptor - SRP-R, a docking protein
Translocon - a multisubunit assembly, a protein conducting channel
Signal-recognition particle (SRP) - six proteins and one RNA molecule (cca 300 nucleotides)
Signal sequence – usually 15 – 30 hydrophobic AA at the N-end of the nascent chain
Removed
signal sequence
Free ribosome
Soluble
protein
Transmembrane
protein
Transport of proteins into the endoplasmic reticulum
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In the membranes of transport vesicles, there are key membrane proteins called
SNARE proteins (soluble N-ethylmaleimide-sensitive-factor attachment protein receptor)
that help draw appropriate membranes together to initiate the fusion process.
These proteins largely determine the compartment with which a vesicle will fuse.
Transport vesicles
Protein-sorting pathways
The mannose 6-P signal in the Nlinked
saccharidic component of
glycoproteins serves as a chemical
marker to target certain lysosomal
enzymes to that organelle.
Integral proteins of the ER
membrane involve at the
carboxylate end the sequence
Lys-Asp-Glu-Leu (KDEL).
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D = aspartate
E = glutamate
K = lysine
L = leucine
R = arginine
S = serine
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Model for the import of nuclear encoded proteins
into the mitochondrial matrix
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Many proteins are synthesized from the mRNA template as a precursor
molecule, which then must be modified to achieve the active protein.
Hydrolytic clipping of proproteins (prohormones, zymogens, etc.)
Examples: The functional insulin molecule is formed by removal of the peptide C
that connects the chains A and B in the proinsulin molecule,
inactive proteinases pepsinogen, trypsinogen, proelastase, etc.
are activated by hydrolysis of small regions of the polypeptide chains,
proopiomelanocortin may be hydrolyzed to corticotropin, MSH sequences,
endorphins and enkephalins.
Many other posttranslational modifications of proteins occur:
glycosylation in synthesis of glycoproteins,
hydroxylation of proline residues to hydroxyproline (collagen, elastin) or
lysine residues to hydroxylysine (collagen),
γ-carboxylation of glutamate residues in some blood-clotting factors,
acetylation (of histones), methylation, prenylation, etc.
Posttranslational modification of proteins
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Monosaccharides found in saccharidic components of glycoproteins
Abbreviation:
Hexoses: Glucose Glc
Galactose Gal
Mannose Man
Acetyl hexosamines: N-Acetylglucosamine GlcNAc
N-Acetylgalactosamine GalNAc
Pentoses: Xylose Xyl
Arabinose Ara
Deoxyhexose
(Methyl pentose): L-Fucose Fuc
Sialic acids: N-Acetylneuraminic acid NeuNAc
(predominant)
Synthesis of glycoproteins
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Glycosyl donors in glycoprotein synthesis
Before being incorporated into the oligosaccharide chains, monosaccharides are
activated by formation of nucleotide sugars.
The glycosyls of these compounds can be transferred to suitable acceptors (catalyzed by
appropriate transferases).
Glucose 6-P Glucose 1-P UDP-Glucose UDP-Galactose
UDP-Glucuronic acid UDP-Xylose
Fructose 6-P Mannose 6-P Mannose 1-P GDP-Mannose
GDP-L-Fucose
N-Acetylglucosamine 6-P N-Acetylglucosamine 1-P UDP-N-Acetylglucosamine
UDP-N-Acetylmannosamine UDP-N-Acetylgalactosamine
N-Acetylneuraminic acid CMP-N-Acetylneuraminic acid
CTP
UTP
GTP
UTP
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O-linked glycosylation
is posttranslational process that takes place exclusively in the Golgi complex and
which is direct – glycosyls from nucleotide sugars (NuDP-glycoses) are transferred
to side chains of Ser or Thr residues and elongated by other nucleotide sugars.
N-glycosylation of "plasma type" glycoproteins
is both cotranslational and posttranslational in the ER and continues
(as posttranslational one) in the Golgi apparatus.
A large oligosaccharide (GlcNAc)2-(Man)9-(Glc)3 destined for attachment to
a N-glycosylated protein is assembled attached to dolichol phosphate and
subsequently transferred en bloc to the protein acceptor, to a specific asparagine
residue of the growing polypeptide chain.
The large saccharidic component is then "trimmed" and additional sugars are
attached in the Golgi complex to form diverse specific patterns.
Dolichol phosphate (anchored in the membrane of endoplasmic reticulum)
H-[CH2-C=CH-CH2]n-CH2-CH-CH2-CH2-O–P=O
CH3
n = 18-20
CH3 OH
OH
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Flip of the polar part to the luminal surface of the ER membrane,
addition of the four external mannose
and three glucose residues:
Transfer of the saccharidic component from dolichol-P-P
to an asparaginyl residue of the growing polypeptide chain.
The assembly of dolichol-P-P-(GlcNAc)2(Man)5
on the cytoplasmic surface of the ER membranes:
Dolichol-P
Dolichol-P-P←GlcNAc←GlcNAc
Dolichol-P-P←(GlcNAc)2←(Man)5
Dolichol-P-P←(GlcNAc)2 (Man)9
Dolichol-P-P←(GlcNAc)2 (Man)9 (Glc)3
2 UDP-GlcNAc
5 GDP-Man
4 Dolichol-P-Man
3 Dolichol-P-Glc
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Trimming and final processing of N-linked glycoproteins:
(Glc)3 (Man)9 (GlcNAc)2-protein
– 3 Glc
(Man)9 (GlcNAc)2-protein
(Man)5 (GlcNAc)2-protein
(Man)3 (GlcNAc)2-protein
pentasaccharide core region
High-mannose
N-linked glycoproteins
up tp 4 Man
removed
– 2 Man
glycosylation
Hybride types
Complex type
N-linked glycoproteins
glycosylation
Late phase processing
Trimming
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Complex type
(triantennary chain)
Hybride type
(one antenna)
High-mannose type
(before trimming to core region)
Examples of plasma-type (N-linked) oligosaccharides
The boxed area encloses the pentasaccharide core region common to all N-linked
glycoproteins.
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Schematic pathway of oligosaccharide processing in newly synthesized glycoproteins:
The coloured symbols represent
N-acetylglucosamine
mannose
glucose
fucose
galactose
N-acetylneuraminic acid
Dolichol
Cotranslational N-glycosylation
to lysosome
Targeting
Processing - early phase (trimming)
Processing - late phase
Finishing by sialylation