Protein Synthesis
Outline Central dogma Genetic code Ribosome Structure and Assembly Mechanics of Protein Synthesis Protein Synthesis in Eukaryotes Inhibitors of Protein Synthesis Postranslation modification of protein
DNA is the genetic material within the nucleus. Central Dogma The process of replication creates new copies of DNA. DNA Replication The process of transcription creates an RNA using DNA information. RNA Transcription Nucleus The process of translation creates a protein using RNA information. Protein Translation Cytoplasm
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
The genetic code 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 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 synthesis require 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 Mg 2+ 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-trna f Met It is only used for initiation, and regular Met-tRNA m Met 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 ribosomebinding site - is known as the Shine- Dalgarno sequence Initiation factor proteins, GTP, N-formyl-MettRNAfMet, 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-trna f Met IF-2 delivers the initiator trna in a GTPdependent 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 trna i Met but it is not formylated
Eukaryotic Initiation Begins with formation of ternary complex of eif-2, GTP and Met-tRNA i Met 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-tRNA i 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 Synthesis Two 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 Secondary structure Tertiary structure Quaternary structure sequence of amino acids shapes formed with regions of the protein (helices, coil, sheets) shape of entire folded protein due to interactions between particular peptides 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) Methylation undergo 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 Acetyl CoA + H 2 N-protein Acetyl-NH-protein + CoA (donor of acetyl-group) 14- C myristoyl CoA + H 2 N-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 T 4 -tyroxine and T 3 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 T 4 and T 3.