Protein Synthesis - TOP Recommended Websites · Cytoplasm Nucleus DNA DNA is the genetic material...
Transcript of Protein Synthesis - TOP Recommended Websites · Cytoplasm Nucleus DNA DNA is the genetic material...
Protein
Synthesis
• Central dogma
• Genetic code
• Ribosome Structure and Assembly
• Mechanics of Protein Synthesis
• Protein Synthesis in Eukaryotes
• Inhibitors of Protein Synthesis
• Postranslation modification of protein
Outline
Cytoplasm
Nucleus
DNA
DNA is the genetic material
within the nucleus.
Central Dogma
RNA
Protein
Replication
The process of replication
creates new copies of DNA.
TranscriptionThe process of transcription
creates an RNA using
DNA information.
TranslationThe process of translation
creates a protein using
RNA information.
GENES
A gene may consist of hundreds or thousands of
nucleotides
Genes are regulated by the degree of coiling
Genes that are tightly coiled can not be activated to make
proteins
Prior to activation the DNA containing the gene of interest
must unwind
Once the molecule has unwound, the enzyme RNA
polymerase can bind to the initial segment of the gene
and protein synthesis can begin
A gene does not build proteins directly, instead it
dispatches instructions in the form of RNA which
programs protein synthesis
Virtually all organisms share the same
genetic code
All organisms use the same 20 amino acids
Each codon specifies a particular amino acid
Trp and Met have only 1 codon each
All the rest have more than one
AUG has a dual function
3 stop codons that code for termination of
protein synthesis
The genetic code
The genetic code has always been believed to be
universal
All known organisms use the same genetic
code. The same codons in the mRNA signal
for the same AAs in plants, bacteria, fish,
frogs, monkeys and humans
The genetic code is degenerate
For most amino acids, there is more then 1
codon or triplet Because of this, the genetic
code is said to be degenerate e.g. GGU,
GGC, GGA, and GGG all encode glycine. The
first two bases alone specify the amino acid
The genetic code is referred to as wobble
The third position of the codon can contain
any of the 4 normal bases (A, G, C, U)
This relative nonspecificity of the third base
of the genetic code is referred to as wobble
Wobble occurs in the codons for many of the AAs.
Genetic code is commaless,
no punctual signal is required to indicate
the end of the codon and the beginning of the text
Genetic code is nonoverlaping,
each base of the triplet is used only once
for the corresponding polypeptide
and the triplets do not overlap
Protein synthesisrequire the functioning of all three major classes of RNA.
• The directions are given by mRNA with each
three-base sequence serving as a codon for a
single AA
• tRNA and the aminoacyl tRNA synthetases
serve as the translator of the language of AAs
and that of nucleotides
• Ribosome provide the enzymes and the
structure on which the entire process takes
place
MECHANISM OF PROTEIN
SYNTHESIS
Like RNA synthesis, protein synthesis or translation
can be divided into stages:
Activation (Preinitiation)
Translation
ACTIVATION (PREINITIATION)
The activation phase of protein synthesis involves
the binding of AA to a specific RNA
The reaction is catalyzed by enzymes called
aminoacyl-tRNA synthetases
These enzyme must recognize both
- specific AA
- its correponding tRNA
and be very specific in their interaction
Because there are 20 AAs that occur naturally in
protein, there must be
at least 20 different amino acyl tRNA synthetases
Aminoacyl-tRNA Synthetase
– one for each amino acid
– 2 step mechanism
– attachment AA to AMP
– transfer to 3’ (or 2’ and then rearrange)
– proofreading function
– can remove an AA incorrectly added to the tRNA
Preinitiation - Charging the tRNA
In the first step of the activation reaction,
the synthetase enzyme attaches the AA to the AMP
portion of ATP with the hydrolysis of pyrophosphate
to form aminoacyladenylate (aminoacyl-AMP)
In the second reaction,
the AA is transferred to either the 2 or 3 -OH of the adenosine
on the 3 end of appropriate tRNA
This process is reffered as charging of the tRNA
Ribosome Structure and
Assembly• E. coli ribosome is 25 nm diameter, 2520 kD in
mass, and consists of two unequal subunits that dissociate at < 1mM Mg2+
• 30S subunit is 930 kD with 21 proteins and a 16S rRNA
• 50S subunit is 1590 kD with 31 proteins and two rRNAs: 23S rRNA and 5S rRNA
• These ribosomes and others are roughly 2/3 RNA
• 20,000 ribosomes in a cell, 20% of cell's mass
Ribosome
Assembly/Structure
• If individual proteins and rRNAs are mixed,
functional ribosomes will assemble
• Gross structures of large and small
subunits are known – see next figure
• A tunnel runs through the large subunit
• Growing peptide chain is thought to thread
through the tunnel during protein synthesis
Eukaryotic Ribosomes
• Mitochondrial and chloroplast ribosomes are quite similar to prokaryotic ribosomes, reflecting their supposed prokaryotic origin
• Cytoplasmic ribosomes are larger and more complex, but many of the structural and functional properties are similar
Mechanics of Protein
Synthesis• All protein synthesis involves three phases:
initiation, elongation, termination
• Initiation involves binding of mRNA and initiator
aminoacyl-tRNA to small subunit, followed by
binding of large subunit
• Elongation: synthesis of all peptide bonds - with
tRNAs bound to acceptor (A) and peptidyl (P)
sites.
• Termination occurs when "stop codon" reached
Prokaryotic Initiation
• The initiator tRNA is one with a formylated
methionine: f-Met-tRNAfMet
• It is only used for initiation, and regular
Met-tRNAmMet is used instead for Met
addition
• N-formyl methionine is first aa of all E.coli
proteins, but this is cleaved in about half
• A formyl transferase adds the formyl group
More Initiation• Correct registration of mRNA on ribosome
requires alignment of a pyrimidine-rich sequence on 3'-end of 16S RNA with a purine-rich part of 5'-end of mRNA
• The purine-rich segment - the ribosome-binding site - is known as the Shine-Dalgarno sequence
• Initiation factor proteins, GTP, N-formyl-Met-tRNAfMet, mRNA and 30S ribosome form the 30S initiation complex
Events of Initiation• 30S subunit with IF-1 and IF-3 binds
mRNA, IF-2, GTP and f-Met-tRNAfMet
• IF-2 delivers the initiator tRNA in a GTP-
dependent process
• Loss of the initiation factors leads to
binding of 50S subunit
• Note that the "acceptor site" is now poised
to accept an incoming aminoacyl-tRNA
The Elongation Cycle• The elongation factors are vital to cell function,
so they are present in significant quantities (EF-Tu is 5% of total protein in E. coli
• EF-Tu binds aminoacyl-tRNA and GTP
• Aminoacyl-tRNA binds to A site of ribosome as a complex with 2EF-Tu and 2GTP
• GTP is then hydrolyzed and EF-Tu:GDP complexes dissociate
• EF-Ts recycles EF-Tu by exchanging GTP for GDP
Peptidyl Transferase
• This is the central reaction of protein synthesis
• 23S rRNA is the peptidyl transferase!
• The "reaction center" of 23S rRNA is shown in next Figure - these bases are among the most highly conserved in all of biology.
• Translocation of peptidyl-tRNA from the A site to the P site follows
The Role of GTP Hydrolysis
• Three GTPs are hydrolyzed for each
amino acid incorporated into peptide.
• Hydrolysis drives essential conformation
changes
• Total of five high-energy phosphate
bonds are expended per amino acid
residue added - three GTP here and
two in amino acid activation via
aminoacyl-tRNA synthesis
Peptide Chain Termination
• Proteins known as "release factors"
recognize the stop codon at the A site
• Presence of release factors with a
nonsense codon at A site transforms
the peptidyl transferase into a
hydrolase, which cleaves the peptidyl
chain from the tRNA carrier
Eukaryotic Protein
Synthesis
• Note the 5'-methyl-GTP cap and the poly A tail
• Initiation of protein synthesis in eukaryotes
involves a family of at least 11 eukaryotic
initiation factors
• The initiator tRNA is a special one that carries
only Met and functions only in initiation - it is
called tRNAiMet but it is not formylated
Eukaryotic Initiation• Begins with formation of ternary complex of eIF-2,
GTP and Met-tRNAiMet
• This binds to 40S ribosomal subunit:eIF-3:eIF4C complex to form the 40S preinitiation complex
• Note no mRNA yet, so no codon association with Met-tRNAi
Met
• mRNA then adds with several other factors, forming the initiation complex
• Note that ATP is required!
• Proteins of the initiation complex apparently scan to find the first AUG (start) codon
Regulation of Initiation
Phosphorylation is the key, as usual
• At least two proteins involved in initiation (Ribosomal protein S6 and eIF-4F) are activated by phosphorylation
• But phosphorylation of eIF-2a causes it to bind all available eIF-2B and sequesters it
Inhibitors of Protein SynthesisTwo important purposes to biochemists
• These inhibitors have helped unravel the mechanism of protein synthesis
• Those that affect prokaryotic but not eukaryotic protein synthesis are effective antibiotics
• Streptomycin - an aminoglycoside antibiotic -induces mRNA misreading. Resulting mutant proteins slow the rate of bacterial growth
• Puromycin - binds at the A site of both prokaryotic and eukaryotic ribosomes, accepting the peptide chain from the P site, and terminating protein
synthesis
Diphtheria Toxin
An NAD+-dependent ADP ribosylase
• One target of this enzyme is EF-2
• EF-2 has a diphthamide
• Toxin-mediated ADP-ribosylation of EF-2 allows it to bind GTP but makes it inactive in protein synthesis
• One toxin molecule ADP-ribosylates many EF-2s, so just a little is lethal!
Ricin from Ricinus communis (castor bean)
• One of the most deadly substances known
• A glycoprotein that is a disulfide-linked
heterodimer of 30 kD subunits
• The B subunit is a lectin (a class of proteins
that binds specifically to glycoproteins &
glycolipids)
• Endocytosis followed by disulfide reduction
releases A subunit, which catalytically
inactivates the large subunit of ribosomes
Ricin A subunit mechanism• Ricin A chain specifically attacks a single,
highly conserved adenosine near position
4324 in eukaryotic 28S RNA
• N-glycosidase activity of A chain removes
the adenosine base
• Removal of this A (without cleaving the RNA
chain) inactivates the large subunit of the
ribosome
• One ricin molecules can inactivate 50,000
ribosomes, killing the eukaryotic cell!
CO-TRANSLATION MODIFICATION OF
PROTEIN
Occurs during synthesis of polypeptide chain. It includes:
1. Proteolytic cleavage -
splitting of Met (+ few more AAs eventually)
by aminopeptidase
2. Tertiary structure formation
3. Disulfide bond formation
4. Group addition
glycosylation, hydroxylation, phosphorylation
of the side chains
FOLDING OF PROTEINS
Includes formation of tertiary and quarternary structure
Proceeds in many steps
1. Small segments with secondary structure
(-helix or -structure of pleeted sheet) are formed,
- account for 8-15 AAs residues. They function like
crystallization centers
2. Growing of segments up to 200 AAs residues
3. Coiling of chains and arrangment to corresponding structure
4. Formation of final conformation.
"In vivo" chapperones are involved in this process
Chapperones are proteins, they can be divided in Hsp 70 Hsp 60
Hsp 70 - recognizes hydrophobic part of nascent protein,
then binds to this structure and prevents unproper
polypetide chain association. Maintances polypetide chain
association only partly folden
Hsp 60 - inside of its structure is vacuole where folding process is
completed
Levels of protein structure
Primary structure sequence of amino acids
Secondary structure shapes formed with regions
of the protein
(helices, coil, sheets)
Tertiary structure shape of entire folded
protein due to interactions
between particular peptides
Quaternary structure structures formed by interaction
of several proteins together
e.g. Functional hemoglobin is
2 -hemoglobin proteins and
2 -hemoglobin proteins
Post-translational Modifications
Follows after protein synthesis termination,
when polypeptide chain is released from ribosome.
It includes:
Partial hydrolysis
from hormone-inactive proinsulin after hydrolysis
link-peptide C insulin
Specific hydrolysis by specific proteinases
Some proteins are synthesized as a segments of polyproteins. Polyprotein in its own
molecule contains sequence of 2-or more proteins. Such way are synthesized many peptide hormone, neurotransmitters
(enkefalins, endorfins), proteins of viruses that are responsible for AIDS…
Glycosylation
Many proteins in ER and Golgi are linked to oligosaccharides to form glycoproteins
In ER proceeds "basic glycosylation"- attachment of oligosaccharides to
Ser or Thr of protein by O-glycosidic bond
In Golgi follows "termination of glycosylation",glycosylation is completed to
different part of cells
Glycosylation - solubility in water
- prevents against hydrolysis by
proteinases
Example: Immunoglobulins
Phosphorylation
catalyzed by protein kinase
The phosphate groups are bound
to the -OH of Ser, Thr, Tyr
in mammals 1000 : 10 : 1
ATP + protein phosphoprotein + ADP
(Casein of milk, histones, many regulatory enzymes)
Methylationundergo Lys, His, Arg of muscle protein +
histones
monomethyllysine
dimethyllysine occur in cytochrome "C"
trimethyllysine occurs in calmodulin
Acylation
Occurs mostly in histones, but also in other proteins.
The amino-terminal bond of protein
AcetylCoA + H2N-protein Acetyl-NH-protein +
CoA (donor of acetyl-group)
14- C myristoylCoA + H2N-protein (GAG,
(donor of 14 C atoms) pol proteins of HIV 1)
Prenylation
Is transfer of 15 C from farnesyl-P-P }
or 20 C from geranyl-geranyl-P-P } to proteins
farnesyl-P-P Transducin (G-protein)
geranyl-geranyl-P-P -subunit of G-protein
Sulphation
Sulphate group is covalently bound
to -OH group of Tyrosine.
The reaction occur in "Golgi".
As a donor of sulphate group serves PAPS.
Iodation
Biosynthesis
of thyroid hormones T4 -tyroxine and T3
ocurs as iodation of tyrosyl residues
of thyreoglobulin
(not by iodation of free Tyr residue and then
followed condensation).
Thyreoglobulin then undergoes degradation
by catepsins in lysosomes
releasing free T4 and T3.