PPT Protein Synthesis

Slide 1 : PROTEIN SYNTHESIS 15-10-2011 5.30 PM By Dr. Ichha Purak http://www.dripurak.com http://www.drichhapurak.webnode.com 10/14/2011 Protein Synthesis 1

Slide 2 : WHAT ARE PROTEINS MECHANISM OF PROTEIN BIOSYNTHESIS REQUIREMENTS FOR PROTEIN SYNTHESIS STEPS IN PROTEIN SYNTHESIS ACTIVATION OF AMINO ACID LOADING OF AMINO ACID TO TRANSFER RNA INITIATION OF POLYPEPTIDE CHAIN ELONGATION OF POLYPEPTIDE CHAIN PEPTIDE BOND FORMATION TERMINATON OF POLYPEPTIDE CHAIN POST TRANSLATIONAL MODIFICATION OF PPC TARGETTING OF PROTEINS 10/14/2011 Protein Synthesis 2

Slide 3 : WHAT ARE PROTEINS Proteins are macromolecules with high molecular weight and are polymers of Amino Acids. Proteins are complex polypeptides having more than 50 Amino Acids and high molecular weight Proteins are the main building blocks and are very important functional molecules of the cell. Almost 20% of a eukaryotic cell’s weight is of proteins. Proteins are widely used in cells to serve diverse functions. Some proteins provide the structural support for cells while others act as enzymes to catalyze certain reactions and some help in transport across membranes 10/14/2011 Protein Synthesis 3

Slide 4 : Important functions performed by proteins are Proteins provide structural frame work of the cell All membranes are of lipo-proteinous nature Proteins maintain cell volume and osmotic balance of the cell Act as biocatalyst (Enzymes) ,regulate the metabolism Help in storage of some elements Act as oxygen carrier ( Haemoglobin ,leghaemoglobin ) Form colloidal system of Protoplasm Act as storage proteins (seeds of cereals,pulses ) 10/14/2011 Protein Synthesis 4

Slide 5 : MECHANISM OF PROTEIN BIOSYNTHESIS Protein biosynthesis is the process in which cells polymerize Amino Acids. It is a multi step process involving amino acid synthesis, transcription of nuclear DNA into messenger RNA followed by translation or polypeptide synthesis . Transcription and Translation are two aspects of Flow of information termed as central dogma of Molecular biology The precise content, and the sequence of amino acids, of a specific protein, is determined by the sequence of the bases in the gene that encodes that protein. The order and chemical properties of the linked amino acids determine the structural organization and biological activity or function of the protein. Protein biosynthesis although very similar, differs between prokaryotes and eukaryotes 10/14/2011 Protein Synthesis 5

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Slide 7 : 10/14/2011 Protein Synthesis 7 CENTRAL DOGMA

Slide 8 : The incorporation of a particular amino acid in the correct position is wonderfully controlled and directed by a template or message ,which starts right from a segment of a strand of DNA (Gene ) . The gene transcribes mRNA which carries the message or information ( as genetic code ) of DNA to the site of Protein synthesis that is ribosome present in cytoplasm. The genetic code is the total sum of codons present on mRNA. Each codon consists of sequence of 3 bases. In the ribosome the message carried by mRNA is translated with the help of a number of loaded tRNA A tRNA possesses a anticodon (3 base sequence complementary to the codon ) by which it can recognize a particular codon on mRNA as well as a particular Amino Acid and can introduce Amino acid at a specific place in the growing polypeptide chain. 10/14/2011 Protein Synthesis 8

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Slide 10 : REQUIREMENTS FOR PROTEIN SYNTHESIS Protein Synthesis Machinery involves Activated Amino Acids tRNAs mRNA Ribosome (site of protein synthesis) Enzymes & coenzymes (ATP,GTP) Numerous initiation , elongation and release factors. Note : Brief information about the components and processes assisting protein synthesis are included in a separate PPT Presentation 10/14/2011 Protein Synthesis 10

Slide 11 : Protein Synthesis or Translation

Slide 12 : STEPS IN PROTEIN SYNTHESIS ACTIVATION OF AMINO ACIDS Amino Acids which are going to form proteins are selected and brought to high energy level by the enzyme aminoacyl tRNA synthetase. High energy penultimate PO4 bond of ATP reacts with COOH group of Amno Acid and result in formation of Amino Acyl Adenylate which is extremely energetic and reactive Amino Acid (1-20 ) + ATP+Enzyme (1-20) →→→ Amino Acid AMP Enz complex(1-20) + Pyrophosphate 10/14/2011 Protein Synthesis 12

Slide 13 : Activation of Amino Acid by ATP to form Amino Acyl adenylate (Highly energetic) 10/14/2011 Protein Synthesis 13

Slide 14 : FORMATION OF AMINOACYL tRNA COMPLEX Amino Acid is transferred from Amino acyl AMP Enzyme complex to 2’ or 3’ hydroxyle position of ribose of Adenine at CCA end of particular tRNA . An ester bond of high energy nature is formed between COOH group of Amino Acid and OH of Ribose AA-AMP-enz complex + tRNA (1-20) →→→ AAtRNA complex (1-20)+AMP+Enz (1-20) O=C-OH (AA) + OH(tRNA) → O=C- O (Ester linkage ) 10/14/2011 Protein Synthesis 14

Slide 15 : Attachment of a specific amino acid to its corresponding tRNA by aminoacyl acyl-tRNA synthetase 10/14/2011 Protein Synthesis 15

Slide 16 : Loading of Activated Amino Acid to tRNA by formation of ester bond between COOH of AA and OH of Ribose of Adenine of tRNA Ester bond 10/14/2011 Protein Synthesis 16

Slide 17 : FORMATION OF POLYPEPTIDE CHAIN It involves translation by participation of mRNA ( carrying codons ), loaded tRNA carrying specific Amino Acids and anticodon, ribosome and enzymes, co-enzymes and several protein factors. mRNA carries genetic code as sequence of codons ,each as triplet of bases, is single stranded,its message is read in 5’→3’ direction and is short lived Genetic code is universal, degenerate, commaless , non-overlapping and non-ambiguousas It has initiator codons as AUG and GUG which code for Methionine and formyl Methionine and Termination codons as UAA,UAG and UGA. The genetic code has 61 codons recognized by different Amino Acids. Some Amino Acids have more than one Codon (Degenerate) 10/14/2011 Protein Synthesis 17

Slide 18 : DNA mRNA 10/14/2011 Protein Synthesis 18

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Slide 20 : INITIATION OF POLYPEPTIDE CHAIN Steps Translocation of mRNA and ribosomes to cytoplasm in case of Eukaryotes. Prokaryotic cell do not need translocation In Prokaryotes Transcription and Translation are coupled Binding of mRNA to smaller subunit ( 30S or 40S) of Ribosome in presence of IF3 or eIF3 which prevents association of 2 ribosomal subunits, mRNA can only bind to dissociated smaller subunit. Diagram on Next Slide 10/14/2011 Protein Synthesis 20

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Slide 22 : Binding of mRNA in correct position is helped by single rRNA of smaller subunit , which has a short sequence of pryrimidine nucleotides near its 3’ end which establishes H bonds with short sequence of purine nucleotides ( Shine Dalgarno Sequence in Prokaryotes) near 5’ end of mRNA near Initiation codon. As a result mRNA-30S-IF3 or mRNA-40S-eIF3 complex is formed. During this complex formation mRNA is so oriented that it faces its initiator codon opposite to P (peptidyl site) of smaller sub unit of ribosome and 2nd codon to A (Acceptor or Active site ) . These two sites are sort of working tables. 10/14/2011 Protein Synthesis 22

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Slide 24 : Complimentary binding between Prokaryotic Shine Dalgarno sequence of mRNA and 16S rRNA 10/14/2011 Protein Synthesis 24

Slide 25 : As a result mRNA-30S (40 S)-tRNA-fMet-GTP complex is formed and IF3 or eIF3 is released Initiator tRNA Met can bind only to P site because at this mRNA displays initiator codon specific for formyle methionine (Prokaryotes) and Methionine (Eukaryotes). GTP helps in establishing H bonds between Initiator codon and Anticodon. Next step is binding of Initiator tRNA- fMet to the peptidyle site ( P ) of smaller subunit. This step is helped by IF1 and IF2 ( eIF1+eIF2) and also GTP. Note : This is the only tRNA brought to P site all successive aminoacyl –tRNA s will be brought to the A site for peptide elongation. 10/14/2011 Protein Synthesis 25

Slide 26 : To the 30S (40S) –mRNA –tRNA (f) or(I) Met GTP complex larger subunit (50S or 60S) gets associated and two initiation factors IF1 and IF2 or eIF1 and eIF2 are released and GTP is hydrolysed to GDP & Pi. With this initiation complex formation is completed possessing Met tRNA( f ) or (I ) Met at the P site ,bound by H pairing to Initiator codon and adjacent codon (2nd ) of mRNA facing A site ,ready to receive next loaded tRNA whose anticodon is complementary to second codon. 10/14/2011 Protein Synthesis 26

Slide 27 : Steps in Formation of Initiation Complex Formation in Prokaryotes using mRNA ,30S .Initiator tRNAfMet, 50S & IF3& Initiator Codon AUG & GTP 10/14/2011 Protein Synthesis 27 GTP

Slide 28 : FOOT NOTE In Prokaryotes there are two types of tRNA for loading Methionine, one is responsible for transferring Methionine at an intercalary position and other transfers formyle Methionine at the Initiation (start) position. For this Methionine after being loaded to tRNA gets formylated by receiving formyl group from N10formyle tetra hydro folic acid (THFA) catalysed by specific enzyme. Methionine - CH3-S-CH2-CH2CHNH2COOH Formyle Methionine – CH3-S-CH2-CH2-CH-NHCHOCOOH In Eukaryotes also there are 2 types of tRNA for carrying Methionine ,either at start or Intercalary position Prokaryotes Met -tRNAm Eukaryotes Met tRNA mMet Met- tRNA fMet Met tRNAiMet 10/14/2011 Protein Synthesis 28

Slide 29 : Generation of the initiator N-formyl-methionyl-tRNA (fMet-tRNA) 10/14/2011 Protein Synthesis 29

Slide 30 : Complementary antiparallel binding of the anticodon for methionyl-tRNA ( UAC) to the mRNA codon (AUG) for methionine 10/14/2011 30 Protein Synthesis

Slide 31 : mRNA attached to ribosome and aminoacyl tRNAs at P & A sites in the cytoplasm of eukaryotic cells 10/14/2011 Protein Synthesis 31

Slide 32 : ELON GATION OF POLYPEPTIDE CHAIN STEPS It begins with arrival of a new loaded tRNA at A site of Initiation complex whose anticodon is complementary to the 2nd codon of mRNA which faces A site of ribosome. The first or Initiator tRNA at P site becomes the donar tRNA and donates its Amino Acid to tRNA bound at Acceptor site by H bonds between anticodon and codon. First peptide bond is formed between COOH group of first Amino Acid and Amino group of second amino acid. Binding of 2nd tRNA to A site requires GTP and 2elongation factors EFTU & EFTS (eEFU & eEFS). Formation of peptide bond is catalysed by peptidyl transferase enzyme ( a protein associated with larger subunit) 10/14/2011 Protein Synthesis 32

Slide 33 : 10/14/2011 Protein Synthesis 33 Peptidyle transferase enzyme was previously considered as protein enzyme of larger subunit of Ribosomes. Harry Nollar and his colleagues (1992 ) discovered that it is not protein but ribosomal RNA (23S) of larger sununit of ribosome hence it is not protein enzyme but RNA enzyme or Ribozyme

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Slide 37 : After first peptide bond formation first tRNA at P site become deacylated ( Uncharged) and moves to E (Exit ) site and then to cytoplasm to pick up another molecule of Methionine for initiating another polypeptide chain using same messenger RNA. Two Amino Acids are linked to 2nd tRNA at A site. As soon as first tRNA is released the ribosome moves by one codon in 5’ → 3’ direction of mRNA so that its P site now faces the 2nd codon of mRNA and A site faces the 3rd codon of mRNA. So automatically at P site is now 2nd tRNA containing dimer and A site facing 3rd codon is ready to receive next loaded tRNA with complementary anticodon For this translocation EFG(Translocase) + GTP are required. GTP is hydrolysed for each shift. Energy is required for shifting and releasing factors EFU & EFS. 10/14/2011 Protein Synthesis 37

Slide 38 : 3rd tRNA takes position by establishing H bonds with the help of EFU & EFS and GTP.Now 2nd tRNA with dimer becomes donar tRNA (P site ) and 3rd tRNA (Asite) become Acceptor tRNA A second peptide linkage is established between COOH group of 2nd AA and NH2 group of 3rd Amino Acid 2nd tRNA become deacylated and released.Ribosome again shifts by one codon and next elongation step takes place. In this way elongation continues by shifting of ribosome codon by codon in 5’ → 3’direction and insertion of loaded tRNA having complementary anticodon till a stop signal is reached 10/14/2011 Protein Synthesis 38

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Slide 42 : TERMINATION OF POLYPEPTIDE CHAIN Termination of polypeptide chain involves two distinct processes Recognition of termination codon Hydrolysis of peptidyl tRNA on the P site of ribosome Peptide chain elongation continues till the ribosome moving codon by codon along mRNA molecule reaches the point where a termination codon occupies the A site. There are three termination codons UAG,UAA and UGA. Since no tRNA has complementary anticodon to termination codon, there is no aminoacyl tRNA to occupy the A site, which becomes free after the translocation step of previous elongation cycle. 10/14/2011 Protein Synthesis 42

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Slide 44 : The termination codon at the A site is recognized by one of the two protein release factors RF1 & RF2. RF1 recognises UAG and UAA. RF2 recognises UAA & UGA. In eukaryotes a third release factor RF3 helps the factors RF1 & RF2 in recognizing the termination codon . The release factor and GTP bind to the termination codon and this binding apparently alters the specificity of peptidyl transferase enzymes of 50 S subunit such that instead of catalyzing peptide bond formation it catalyses the hydrolysis of ester bond between COOH group of last Amino Acid and OH group of Ribose of Adenine of tRNA at P site carrying polypeptide chain..So the polypeptide chain is released. 10/14/2011 Protein Synthesis 44

Slide 45 : Hydrolysis of GTP leads to dissociate release factors from ribosome following which mRNA and Ribosome subunits are released. Once the tRNA moves from P site, 70S ribosome dissociates into 30S & 50S subunits.mRNA is also released. These subunits can participate in next round of polypeptide synthesis in presence of IF3 POST TRANSLATIONAL MODIFICATIONS OF POLYPEPTIDE CHAIN In prokaryotes either formylation of Methionine or Methionine it self is removed. Aminopeptidases may even remove some AAs from Amino terminus. In eukaryotes Initiating Amino Acid Methionine is often removed when 15-30 AAs have been polymerized. 10/14/2011 Protein Synthesis 45

Slide 46 : Sugar residue may be added to side chain of Aspargine forming glycoproteins AAs like Thr,Ser & Tyr may be phosphorylated by ATP The polypeptide chain starts acquiring secondary and tertiary structure while translation is proceeding. Changes take place continuously through out synthesis until the final product possesses a structure of minimum energy with most polar groups exposed to acquous environment and most non polar groups in the interior of the molecule. 10/14/2011 Protein Synthesis 46

Slide 47 : Loss of Signal Sequence : Some proteins have 15-30 residue of hydrophobic amino acids forming signal sequence which has a role in directing protein to ultimate destination. This signal sequence may be removed while translation or after translation in lumen of Endoplasmic reticulum. Signal sequence is formed due to presence of signal codons in some messenger RNAs just after I codon Glutamic acid and Aspartic acid may change to Glutamine and Aspargin Disulphide bridges may be formed between two cysteine residues of same or different polypeptide chains 10/14/2011 Protein Synthesis 47

Slide 48 : SIGNAL CODONS AND PROTEIN TARGETTING In Eukaryotes Some messenger RNAs possess signal codons just after the Initiator codon on the 5’end terminity. Because of the signal codon, a signal sequence appears at the amino terminus of growing peptide chain The signal sequence is recognised by Signal Recognition Particle (SRP) . Translation is blocked until SRP helps binding of Ribosome to Receptor or Docking Protein present on the membrane of Endoplasmic Reticulum. SRP is dissociated and recycled Polypeptide synthesis resumes and continues on the ribosome bound to E R 10/14/2011 48 Protein Synthesis

Slide 49 : Polypeptide enters inside the lumen of E R through a pore and synthesis is completed there only. These are secretory proteins . From E R lumen these are transported to Golgi apparatus, Lysosome etc and also to exterior of Plasma membrane. Signal sequence of most of the proteins after synthesis or liberation are removed by signal peptidases. Those mRNAs which donot contain the signal codon complete protein synthesis after binding to ribosome free in the cytoplasm itself and give structural proteins. 10/14/2011 49 Protein Synthesis

Slide 50 : Schematic diagram showing the mechanism by which signal sequence directs the destiny of protein through lumen of Endoplasmic Reticulum 10/14/2011 50 Protein Synthesis

Slide 51 : Signal Sequence directing Protein Targetting 10/14/2011 51 Protein Synthesis

Slide 52 : Protein synthesis on free ribosomes or on ribosomes attached to E R membrane 10/14/2011 52 Protein Synthesis

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Slide 54 : SUMMARY OF THE CLASS : PROTEIN SYNTHESIS Protein Biosynthesis is a multistep process It involves polymerisation of 20 Amino Acid by formation of peptide bond between COOH group of one Amino Acid and NH2 group of other Amino Acid The precise content, and the sequence of amino acids, of a specific protein, is determined by the sequence of the bases in the gene that encodes that protein. The message of DNA is transmitted through formation of mRNA by the process known as transcription 10/14/2011 Protein Synthesis 54

Slide 55 : The mRNA carries the message in the form of RNA codons (Triplet of bases ) Specific for a particular amino Acid to the place of protein synthesis i.e. Ribosome present in cytoplasm The first step in protein synthesis is attachment of mRNA to the smaller sub unit of ribosome helped by IF3 mRNA is so oriented that its Initiator codon faces the Peptidyle site and 2nd codon to Acceptor site of Ribosome Amino Acids which take part in protein synthesis are activated by ATP and loaded to Particular tRNA After this initiator tRNA carrying formyl Methionine (Pro) and Methionine (Eu) binds to initiator codon of mRNA by H bonds between codon and Anticodon is helped by IF1 & IF2 10/14/2011 Protein Synthesis 55

Slide 56 : After this larger subunit of ribosome binds to complete formation of Initiation complex which has P & A sites as working tables with 2nd codon ready to accept incoming 2nd tRNA having complementary anticodonon As 2nd tRNA binds to 2nd codon with the help of EFU & EFS, 1st tRNA loaded with Methionine becomes donar tRNA and a peptide bond (C──N) is formed between COOH of Met and NH2 of 2nd Amino acid with release of a molecule of water . The reaction is catalysed by peptidyl transferase enzyme a component of larger sub unit of ribosome. The dipeptide is now bound to 2nd tRNA and first tRNA becomes uncharged. It moves from P site to E (Exit) site. 10/14/2011 Protein Synthesis 56

Slide 57 : The ribosome moves by one codon in 5’→ 3’ direction of mRNA by EFG(Translocase ) and GTP By this movement of ribosome the P site now faces 2nd codon and A site 3rd codon is ready to receive next loaded tRNA with complemetary anticodon 3rd tRNA takes position by making H bonds between codon and anticodon, 2nd tRNA carrying 2 Amino Acids become donar tRNA . 2nd peptide bond is formed between 2nd and 3rd Amino Acid. The process continues till a stop codon is reached on mRNA facing A site. Since no tRNA has complementary anticodon to termination codon (UAG/UAA/UGA) ,so A site remains free after translocation step of previous cycle 10/14/2011 Protein Synthesis 57

Slide 58 : The termination codon at A site is recognised by one of the release factor This binding alters activity of peptidyle transferase enzyme. It catalyses hydrolysis of ester bond between last amino acid and tRNA . Polypeptide chain is released. Also ribosomal sub units are dissociated. Same mRNA can bind to many ribosomes successively, each ribosome translating similar polypeptide chains only differing in length. The structure is known as polysome or polyribosome. 10/14/2011 Protein Synthesis 58

Slide 59 : THANK YOU 10/14/2011 Protein Synthesis 59