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.