1 Synthesis of proteins. Posttranslational modifications of proteins. ÓDepartment of Biochemistry Faculty of Medicine (E.T.) 2013 2 Translation – synthesis of proteins Which cells: all cells having nuclear DNA Where in the cell: ribosoms (free or attached to ER) mitochondrias Differences between prokaryotes and eukaryotes: prokaryotes: transcription, processing of transcript and translation are not separated by space eukaryotes: translation starts after the mRNA synthetized in nucleus is transported to cytoplasma 3 3 Molecules and other species necessary for synthesis of proteins Species Funkce Amino acids Substrates for proteosynthesis Many enzymes Catalysts Protein factors efectors ATP, GTP energy Inorganické ions (Mg2+, K+) Cofactors of enzymes tRNA Transfer of AA to ribose mRNA Determines the order of AA in a protein rRNA Structural role, catalyzes the formation of peptide bond 4 Phases of translation A.Inciation B.Elongation C.Termination They occur in cytoplasma, in connection with ribosomes 5 Genetic code Proteins – 20 different AA RNA – 4 bases Each amino acid is characterized by a triplet of bases in mRNA – codon Codons consisting of three nucleotide can provide 64 variations → 61 of them code for 20 amino acids 3 codons are STOP codons (UAA,UAG, UGA) Nierenberg (1961) – poly(U) sequence of mRNA produced polyphenylalanine by translation Þ sequence UUU is the codon for phenylalanine 6 First base Second base Third base 5´ U C A G 3´ Phe Ser Tyr Cys U Phe Ser Tyr Cys C U Leu Ser Stop Stop A Leu Ser Stop Trp G Leu Pro His Arg U Leu Pro His Arg C C Leu Pro Gln Arg A Leu Pro Gln Arg G Ile Thr Asn Ser U A Ile Thr Asn Ser C Ile Thr Lys Arg A Met Thr Lys Arg G G Val Ala Asp Gly U Val Ala Asp Gly C Val Ala Glu Gly A Val Ala Glu Gly G Genetic codon 7 • it is degenerate (more than one codon can code for an amino acid) • it is unambiguous (each codon specifies one and only one amino acid) • it is almost universal ( the code is identical in prokaryotes and eukaryotes with some exceptions : e.g. human mitochondrial mRNA the triplet UGA codes for trp instead of stop-codonu, AUA codes for methionine instead leucinu) • it can wobble (base pairing between the last nucleotide of the triplet and corresponding nucleotide is not strictly given by Watson-Crick rule) • it is nonoverlapping and „comaless“ (the codons are aligned without overlap and without empty spaces. Each base belongs to one codon and only one codon) Prominent properties of genetic codon: 8 Degeneracy of the genetic code 9 Relation between mRNA and the protein product •Sequence of bases in mRNA is sorted into codons •The start codon (AUG) sets the reading frame •The order of codons in the mRNA determines the linear sequence of amino acids in the protein – the reading is given by reading frame ……A U G C A C A G U G G A G U U………. Only one of the three possible frames is the right one, it begins at the start codon AUG recognized by the Met-tRNAMet > 10 Efect of mutations Mutations are structural alterations that result from damage of DNA molecules or unrepaired errors during replication They can be transcribed into mRNA By translation of altered base an abnomal sequence of amino acids can appear in the protein 11 Types of mutation 1. point mutation ® exchange of single base a) No detectable effect – because of degeneration of the code – silent mutation e.g. CGA®CGG (both sequences code for Arg) b) Missence effect – different amino acid is incorporated at the corresponding site in the protein molecule e.g. GCA®CCA results in replacement of arg by prolin c) nonsense – they result in premature termination e.g. CGA ®UGA, the codon for Arg is replaced by stop-codon 12 Types of mutation (cont.) 2. insertion – one or more nucleotides are added to DNA 3. deletion – one or more nucleotides are removed from DNA The damage of the protein depends on the number of deleted or inserted 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. 13 Example of point mutation Point mutations in the genes for hemoglobin: There is known about 800 of structural variants of human hemoglobin Most of them result from point mutations and are not harmfull. Some of them cause diseases. Methemoglobinemia – e.g. replacement of single histidine by tyrosine in a-chain Þ protein is unattackable for the treatment of methemoglobin reductase, the methemoglobin in blood rises Sickle cell anemia – missense mutation, GTG replaces GAG Þ replacement of glutamate by valine in position 6 of b-chain Þ the chain become less soluble and precipitate when deoxygenated Example of nonsens mutation b0-thalasemia It is caused by mutation on codon 17 in the both allels. The synthesis of the b-chian is early terminated. 14 Amino cids cannot react directly with bases „the adapter molecules are tRNAs“ • each molecule of tRNA contains anticodon • anticodon is a triplet of bases that are complementar with the codon in mRNA • each tRNA can bind specific AA on its 3´-terminal The genetic code defines the relationship between the base sequence of the messenger RNA and the amino acid sequence of the polypeptide: Significance of DNA molecules 15 General structure of tRNA molecules The cloverleaf structure 16 Formation of aminoacyl-tRNA • aminoacid is firstly activated by a reaction of its carboxylic group with ATP to aminoacyl-AMP • activated AA is transferred to 2´- or 3´- OH group of ribose on 3´terminal of tRNA • reaction is catalyzed by specific enzymes (aminoacyl-tRNA synthetases) • 20 different synthetases exist, one for each AA • the cleavage of ATP in the first reaction provides energy • 17 N N N N N H 2 O O H O H O P O O O Aminoacyl-AMP (mixed anhydride) + ATP C O C O H N H 2 R H 2Pi + 1. Activation of AA C C O H N H 2 R 18 2. Transfer of activated AA to 3´-end of tRNA Cyt t-RNA 3´- end of t-RNA Ester bond between -COOH of amino acid and 3´-OH of ribose Amino acid C O C N H 2 R N N N H 2 P O O - N N C H 2 O O H O O P O O O - C H 2 O O H O O 19 (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. Aminoacyl-tRNA synthetases 20 Ribosomes Large ribonucleoprotein particles – composed of proteins and RNA Free floating in the cytoplasma or attached to the membranes Ribosomes are workbenches for protein synthesis 21 Ribosomes Ribosomes consist of a large and a small subunit Inactive ribosomes exist as loose subunits that aggregate into complete particles when they get ready for protein synthesis. Large ribosomal subunit has three binding sites for molecules of tRNA – P, A, E P-peptidyl-tRNA A-aminoacyl-tRNA E-free tRNA (exit) E P A 22 Prokaryotic x eukaryotic ribosomes Property Bacterial Human Sedimentation constants: complete ribosome small subunit large subunit RNA content RNA-small subunit RNA-large subunit Placement in the cell 70S 30S 50S 65% 16S 5S 23S Free floating in cytoplazma or attached to plasmatic membrane 80S 40S 60S 50% 18S 5S 5,8S 28S Free floating in cytoplazma or attached to ER membrane 23 Initiation Ribosome has to associate with mRNA and the initiator tRNA Formation of initiation complex. Soluble cytoplasmic factors help in initiation – initiation factors Also GTP, ATP are involved Differences between eukaryonic and prokaryonic cells 24 Initiation u eukaryotes 5´-P-P-P-5´- 5´ G Small ribosomal subunit Large ribosomal subunit Guanine cap mRNA E P A G Iniciation factors It involves formation of a complex composed of methionyl t-RNAmet, mRNA and a ribosome. anticodon Aminocyl-tRNA tRNA 25 40S eIF3 eIF1A Binding of factors eIF3 and eIF1A to a small subunit > 26 G eIF2 GTP AMP Met 3´ Binding of activated Met to tRNAMet Binding of GTP to eIF2 eIF2 is heterotrimeric G-protein > 27 Met Factor eIF2 recognizes the initiation aminoacyl-tRNA 3´ G GTP eIF2 UAC Binding of complex GTP-eIF2 to t-RNAMet > 28 m-RNA A C A U G U U G C C…... 5´-P-P-P-5´- 5´ G CBP (cap binding protein) binds to the cap on 5´end of mRNA CPB (eIF-4F) is composed of a number of other initiation factors (eIFs) > 29 G GTP eIF2 UAC Met Complex Met-tRNAMet, eIFs and GTP binds to the snaller ribosomal subunit Preiniciation complex > 30 G GTP eIF2 UAC A C C G U A A C A U G U U G C C G 5´-P-P-P-5´- m-RNA Binding of m-RNA to preiniciation complex Reaction requires ATP for unwinding a hairpin loop in the mRNA (helicase activity of an eIF subunit) The complex scans mRNA from 5´end until it locates the AUG start codon > 31 UAC A C C G U A A C A U G U U G C C G 5´-P-P-P-5´- A P E • GTP is hydrolyzed •eIF separates Pi + GDP eIF •The larger ribosomal unit is attached •Met-tRNA bimds at the site P of larger ribosomal subunit Met Iniciation complex 80S > 32 Differences in between prokaryotes and eukaryotes eukaryotes prokaryotes Binding of mRNA to smaller ribosomal subunit Cap on the 5´end of mRNA binds IFs and 40S subunit containing t-RNAmet. mRNA is scanned until AUG No cap, Shine-Dalgarno sequence in mRNA about 10 nucleotides upstream of the AUG start codon is attached to a complementary sequence in 16S RNA First AA methionine formylmethionine Iniciation factors 12 and more 3 ribosomes 80s (40s a 60s) 70s (30s a 50s) 33 eIF2 factor in eukaryotes eIF2 has essential significance for initiation of translation – control point in proteosynthesis It is heterotrimric G-protein, it binds GTP and GDP Its phosphorylated form in inactive – regulation of its activity by action of proteinkionases Conditions such starvation, heat shock, viral infection, glucose starvation result in phosphorylation of eIF2 by specific kinases (generally conditions when the energy expanditure required for synthesis of proteins would be deleterious) Example of regulation: Synthesis of globin in v reticulocytes • In the absence of heme, the eIF2 is phosphorylated, the rate of initiation of globin synthesis decreases • heme acts by inhibiting the phosphorylation of the eIF2 Þ eIF2 is active in the presence of heme and globine synthesis is initiated 34 Elongation of peptide chain • formation of the second aminoacyl-tRNA • binding of an aminoacyl-tRNA to the A site on the ribosome • formation of peptide bond • translocation of peptidyl-tRNA to the site P 35 UAC A P E Antikodon is AAC Amino acids is leucin A C C G U A A C A U G U U G C C G 5´-P-P-P-5´- Which amino acid will be added? The next codon is UUG 36 G GTP EF1a (elongation factor) G GTP EF1a AAC Leu 1.Activation of leucine by reaction with ATP ® leucinyladenylate 2. formation of Leu-tRNA 3. + binding of GTP a EF1a Formation of Leu-tRNA 37 UAC A P E A C C G U A A C A U G U U G C C G 5´-P-P-P-5´- AAC Met Leu Leu-tRNA binds to the site A A P E GTP is hydrolyzed to GDP + Pi, complex GDP-EF1a is released GDP-EF1a Pi Process of elongation is very similar in eukaryotes and prokaryotes (different cofactors of elongation) 38 UAC A P E A C C G U A A C A U G U U G C C G 5´-P-P-P-5´- AAC Met Leu A P E Binding site on tRNAMet is empty Peptidyltransferase is rRNA. It is a component of 28S RNA subunit 60S – ribozyme activity Formation of peptide bond (transpeptidation) Synthesis of proteins starts with N-terminal Peptidyltransferase catalyzes the release of methionine from tRNA and its transfer to leucine. A peptide bond between carboxyl group of methionine and amino group of leucine is formed 39 UAC A P E A C C G U A A C A U G U U G C C G 5´-P-P-P-5´- AAC Met Leu A P E Movement of met-tRNA to the site E 1. EF2 and GTP bind to ribosome EF2 + GTP 2. tRNAMet moves to the site E, site P become free G > 40 A P E A C C G U A A C A U G U U G C C G 5´-P-P-P-5´- AAC Met Leu A P E Result of animation on page 42 UAC G Movement of met-tRNA to the site E 41 A P E A C C G U A A C A U G U U G C C G 5´-P-P-P-5´- AAC Met Leu A P E Translocation and release of Met-tRNA GDP-EF2 + Pi Movement of ribosome with respect to mRNA and its base-paired tRNAs® peptidyl-tRNA moves into the P site Site A is empty GTP is hydrolyzed, EF2 will release Direction of movement > 42 Which further steps will follow? Next step of elongation Further tRNA charged with amino acid (proline) binds to the site A 43 Termination • elongation steps are repeated until a termination (stop) codon moves into the A site of the ribosome • no tRNA that can pair with stop codon is present in cytoplasma • releasing factors bind to ribosome instead • peptidyltransferase hydrolyzes the bond between peptide chain and tRNA • newly synthesized peptide is released from ribosome • ribosome dissociates into individual subunits, mRNA releases 44 Energy consumption Aaminoacyl-tRNA formation ATP® AMP + 2 Pi 2 Binding of aminacyl-tRNA to the site A GTP ® GDP + Pi 1 Translocation of peptidyl-tRNA to the site P GTP ® GDP + Pi 1 Equivalent of ATP 4 ATP 4 ATP are required for synthesis of one peptide bond. Further energy is required for initiation and synthesis of nucleotides. Proteosynthesis rate Prokaryotes ~ 100 peptide bond s Eukaryotes ~100 peptide bonds/min 45 Stimulation of proteosynthesis by insulin CBP (cap binding protein complex) has subunit eIF4E This subunit is blocked by protein 4E-BP Insulin triggers phosphorylation of 4E-BP Phosphorylated 4E-BP looses its afinity to eIF4E ® eIF4E become free for participation in protein synthesis Insulin is anabolic hormone, it stimulates proteosynthesis. It affects the synthesis through the interaction with CBP. 46 Polysomes NH2 NH2 NH2 NH2 As one ribosome moves along the mRNA produsing a polypeptide chain, a second ribosome can bind to the vacant 5´-end of mRNA. Many ribosomes can simultaneously translate a single mRNA, forming a complex known as polysome. A single ribosome covers approximately 80 nucleotides of mRNA. Therefore, ribosomes are positioned on mRNA at intervals of approximately 100 nucleotides. Simultaneous translation of mRNA on more ribosomes 47 Synthesis of proteins in mitochondria •Mitochondria contain 2-10 copies of circular double stranded DNA •The size varies depending on the species •Animal mitochondrial DNA - Mr~ 107 •Codes for rRNA, set of tRNAs and mRNA for several proteins •Proteins synthesized in mitochondrial are a negligible proportion of total proteins of inner mitochondrial membrane but are essential for process of oxidative phosphorylatiob ( a part of complexes I,III,IV and ATP-synthase) •Synthesis of of proteins in mitochondria has many common features with synthesis in prokaryotes (e.g. initiation by formylmethionine, sensitivity to antibiotics) mitochondria2 48 Antibiotics Effect Streptomycine Binds to 30S ribosomal subunit, inhibits formation of initiation complex.Triggers errors in reading frame of mRNA. Tetracycline Binds to 30S ribosomal subunit and inhibits binding of aminoacyl-tRNA to site A Chloramfenikol Binds to 50S ribosomal subunit and inhibits peptidyltransferase Erytromycine Binds to 50S ribosomal subunit and inhibits translocation Puromycine Binds to A-site on ribosome and trigers premature termination Effects of antibiotics on prokaryotic proteosynthesis the differences in proteosynthetic procedure between eukaryotes and bacterias are exploited for clinical purposes some antibiotics inhibits specifically proteins of bacterial ribosomes 49 •Nascent polypeptide chain is transported across ribosomes • Outside of the ribosome the N-terminus of the protein begins to fold (while the C-terminal portion of the protein is still being synthesized by the ribosome) • Folding is the process by which a protein achieves its thermodynamically-stable three-dimensional shape to perform its biological function •Specialized proteins called chaperones assist in the folding. Folding of proteins File:Protein folding schematic.png 50 http://www.nature.com/scitable/content/ne0000/ne0000/ne0000/ne0000/14463077/f3_dobson_nature02261-f 2.2.jpg chaperons Folding of proteins 51 51 •Protein folding is the process by which a protein structure assumes its functional shape or conformation– only one of them is correct and is related to the native •Misfolded proteins can be formed by means of high or low temperature, radiation, oxidative stress, chemicals etc. Folding is affected by chaperons. •Incorrect folding can follow from mutation of a gene • Misfolded proteins are ubiquitinated and degraded in proteasome. •Accumulation of misfolded proteins in cell can occur due to overproduction of proteins, their damage or disfunction. •Accumulation can cause disease, known as amyloid diseases. •The most prevalent one is Alzheimer's disease, Parkinson's disease and Huntington's. •Another diseases are caused by lack of a particular functioning protein, due to its degradation as a consequence of misfolding (e.g. cystic fibrosis (misfolded CFTR protein), Marfan syndrome (misfolded fibrillin), Fabry disease (misfolded alpha galactosidase),) Folding 52 •Proteins stabilizing unfolded or partially folded structures, facilitate correct folding and assembly • Many chaperones are heat shock proteins, that is, proteins expressed in response to elevated temperatures or other cellular stresses. •They binnd mainly to hydrophobic areas of proteins • they are present in mitochindria, cytoplasm, lumen ER Chaperons 53 Targeting of proteins to subcelular and extracellular locations Synthesis of proteins on polysomes in cytosol Proteins remain in the cytosol or enter the organeles (nucleus, mitochondria). They contain amino acid sequence (targeting sequence) that facilitate their transport into a certain organelle Synthesis of proteins on ribosom bound to RER Transport to lysosomes, ER, Golgi complex, cellular membranes or secretion 54 Transport of proteins syntesized on RER RER membrane Signal peptide (ussualy 15-30 hydrophobic amino acids on N-terminl end) Signal recognition particle (SRP) SRP-receptor Signal peptidase Cleaved signal peptide 1. 2. 3 4 5 6 55 1.translation begins in the cytosol 2.As the signal protein emerges from the ribosome, a signal recognition particle (SRP) binds to it and to the ribosome snd inhibits further synthesis of the protein 3.the SRP binds to the SRP receptor in the RER membrane, docking the ribosome on the RER 4.The SRP is released and protein synthesis resumes 5.As the signal peptide moves through a pore into the RER, a signal peptidase removes the signal pepide 6.Synthesis of the nascent protein continues and the completed protein is released into the lumen RER Transport of proteins syntesized on RER 56 Lyzosomes Secretory vesicles Cis Golgi Trans Golgi http://vcell.ndsu.nodak.edu/animations/proteintrafficking/first.htm Transport of proteins syntesized on RER 57 • Proteins syntesized ribosomes attached to RER travel in vesicles to the cis face of the Goolgi complex • here is the sorting center – structural features of proteins determine their direction • some remain in Golgi complex, some return to RER • others bud from trans face of the Golgi complex in vesicles • these vesicles become lysosomes or secretory vesicles depending on their contents • secretory proteins are released from the cells when secretory vesicles fuse with the membranes • proteins with hydrophobic regions embedded in the membrane of secretory vesicles become cell membrane proteins Transport of proteins syntesized on RER 58 Principles of intracellular sorting Example 1: Proteins determined for lysosomes are marked by N-bonded oligosacharides terminated by mannose-6-P Prot-oligosaccharide-mannose-6-P „address“ is recognised by specific membrane receptors in Golgi complex that embedds the protein into the clathrine coated vesicles 59 Example 2: Proteins destinated for ER have sequence Lys-Asp-Glu-Leu on their carboxy terminal lys-asp-glu-leu Proteins are transported back from Golgi complex to ER Principles of intracellular sorting 60 Posttranslational processing of proteins • Methionine removal •ADP-ribosylation •Glycosylation •Fatty acylation •Phosphorylation •Acetylation After protein emerge the ribosome, they may undergo posttranslational modification •Carboxylation •Methylation •Prenylation • Hydroxylation •Sulfatation ad. • 61 Glycosylation of proteins Glycoproteins N-linked carbohydrate chain Involving the amide nitrogen of asparagine They differ in composition of saccharides and and the way of synthesis O-linked carbohydrate chain Involving hydroxyl side chain of serine or threonine N-linkage to GalNAc O-linkage to GalNAc 62 Synthesis of N-glycoproteins The oligosaccharide chain is first assembled on the dolichodiphosphate backbone Dolichol is isoprene (see the synthesis of cholesterol) H-[CH2-C=CH-CH2]n-CH2-CH-CH2-CH2OH CH3 CH3 n=18-20 Dolicholdiphosphate is bonded to membrane of ER. Activated monosaccharides are gradually attached to the terminal phosphate. The oligosaccharide chain is then transferred en bloc to suitable Asn residues of apoglycoproteins during their synthesis on membrane-bound polyribosomes 63 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 Synthesis of oligosaccharide precursor Transfer to protein 64 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 65 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. 66 Synthesis of O-glycoproteins ………Ser……………. ………Ser……………. OH UDP-monosach. UDP O-monosacharide 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 (Thr) (Thr)