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Chapter 17 Protein Synthesis , Folding and Processing mRNA directed protein biosynthesis is also called translation in molecular biology. Why ? Protein biosynthesis is a process to translate information from the nucleotide sequence of an mRNA into the sequence of amino acids of the corresponding specific protein. Section One Components Required for Protein Synthesis The process of protein synthesis is one of the most complex events in the cell. It involves the coordinated participation of over 100 biomolecules. Biomolecules Required in Protein Synthesis mRNA the template tRNAs transferring amino acids ribosome the location of protein synthesis aminoacyl-tRNA synthetases link tRNA and amino acid protein factors ATP and GTP inorganic ions A mRNA works as the template in protein synthesis. the structure of a eukaryote mRNA Cap-----AUG-------------stop codon--------poly A 5’UTR coding region 3’UTR What is the relation between the coding region in mRNA and the amino acid se-quence in the corresponding protein? The Genetic Code The genetic code is the way in which the nucleotide sequence in mRNA ( or DNA ) specifies the amino acid sequence in protein. How ? A, G, U and C are organized into triple-nucleotides called codons. There are 64 ( 4x4x4 ) codons. The collection of the 64 codons makes up the genetic code. The genetic code was deciphered in 1966 by Nirenberg et al. In the genetic code 61 codons specify 20 amino acids. They are sense codons. There are 3 codons, UAA, UAG and UGA, which do not specify any amino acids. They are stop codons ( termination codons, nonsense codons ). 1. Five Features of the Genetic Code Universal The genetic code is used by all species, from prokaryotes to human being. However some deviations are known to occur in mitochondria and some unicellular organisms. 2. Directional The sequence of triple-nucleotide codons is read in the direction of 5’ to 3’. The first codon of coding region of mRNA is almost always AUG, the initiation codon. The last codon of the coding region of mRNA is one of the stop codons. 3. commaless The coding region in mRNA is read in a continuing way without punctuation. If there is insertion or deletion of one or two nucleotide(s) in the coding region of mRNA frameshift mutation occurs. AUG UCG CAA GAU ACG UCC Met Ser Gln Asp Thr Ser AUG CGC AAG AUA CGU CC Met Arg Lys Ile Arg AUG CUC GCA AGA UAC GUC C Met Leu Ala Arg Tyr Val 4. degeneracy Because 61 codons specify 20 amino acids multiple codons must decode the same amino acid. That is called degeneracy. For example Leu, Ser, and Arg each is specified by six different codons. Homework Find out other examples of codon degeneracy 5. wobble There is wobble in the process of codon ( in mRNA ) and anticodon ( in tRNA ) base-pairing. That is the base-pairing does not strictly according to the standard base-pairing rule ( A-U , G-C , T-A ). The wobble base pairs includes G-U, I-A, I-C, and I-U, and often appear in the third codon-anticodon position. 1,2, 3 1,2 3 codon CAU/C AU A/C/U anticodon GU G UC I The wobble base-pairing accounts for codon degeneracy. B tRNA is the tool of transferring amino acid. There are 20 amino acids. There are 61 sense codons. There are about three dozens of prokaryotic and about 50 eukaryotic tRNAs Because of wobble base-pairing caused codon degeneracy 61 sense codons can be red by less than 61 tRNAs It is the aminoacyl-tRNA that participates in the protein synthesis. The aminoacyl-tRNA synthetase catalyzes the linkage of an amino acid to its cognate tRNA. C.Ribosome is the location of protein synthesis A ribosome is composed of two subunits, a a large one and a small one. Prokaryotic S 16SrRNA+Ps 30S L 23SrRNA 5SrRNA +Ps 50S Whole 70S Eukaryotic 18SrRNA+Ps 40S 28SrRNA 5.8SrRNA 5SrRNA +Ps 60S 80S 1. 2. Function of Ribosome To hold mRNA, aminoacyl-tRNA and translation factors in right place for protein synthesis. To catalyze certain chemical reactions in the process of protein synthesis D . Other components for protein synthesis 20 amino acids dozens of tRNAs aminoacyl-tRNA synthetases translation factors ( initiation, elongation, and release factors ) ATP and GTP Mg2+ Section Two Protein Synthesis Takes Place in Five Stages A activation of amino acid and synthesis of aminoacyl-tRNA tRNAs are joined to amino acids to become aminoacyl-tRNA in a reaction called aminoacylation. Special enzymes called aminoacyl-tRNA synthetases carry out the joining reaction which is extremely specific. nomenclature of tRNA and aminoacyl-tRNA a.a. cognate tRNA c. synthetase aminoacyl-tRNA ser ser Ser tRNA ser-tRNA synthetase ser-tRNA leu leu Leu tRNA leu-tRNA synthetase leu-tRNA The aminoacylation reaction is a two-step reaction driven by ATP. The first step is activation of amino acid. AA + ATP + E ------------- AA-AMP-E + PPi The second step is charging tRNA. tRNA + AA-AMP-E ---- AA-tRNA+AMP+E A cognate tRNA becomes attached to the aminoacyl group through an ester bond. The ester bond is formed between the acyl group of the amino acid residue and the tRNA’s 3’-OH. Aminoacyl-tRNA synthetase has proofreading activity. B Formation of Initiation Complex ( in prokaryotes ) Initiation is the assembly of a ribosome with fmet fMet-tRNA on an mRNA molecule. How? 1. IF1 and IF3 bind to a free 30S subunit. This helps to prevent a large subunit binding to it without an mRNA and forming an inactive ribosome. 2. mRNA binds to 30 S subunit by way of mRNA and 16SrRNA interaction. There is SD sequence ( RBS , ribosome binding site ) 8-13 nt upstream of the initiation codon in prokaryotic mRNA which base-pairs with a complementary sequence near the 3’ end of 16SrRNA. 5’ AGGAGGU 3’ SD sequence 3’ UCCUCCA 5’ on 16SrRNA That results in the initiation codon in the P site. 3. With the help of IF2 and GTP the initiator fmet tRNA ( fMet-tRNA )can then bind to the initiation codon ( AUG ) on the mRNA. 4. The 50S subunit can now bind, which displaces IF1, IF2 and IF3, and GTP is hydrolyzed to GDP and Pi. The 70S initiation complex is formed. met fmet There are two types of tRNA s , tRNA and met tRNA . Both of them can link a Met, and form fmet met Met-tRNA and Met-tRNA respectively. fmet The Met residue in Met-tRNA is formylated fmet and fMet-tRNA is formed. fMet fMet-tRNA is the initiator tRNA . It recognizes only the initiation codon and participates in translation initiation. met The Met residue in Met-tRNA will not be modified. It participates in translation elongation. Eukaryotic Translation Initiation met There are also two types of tRNA s in eumet met karyotes, tRNAi and tRNAe . met Both of them can link Met and form Met-tRNAi met and Met-tRNAe . met Met-tRNAi is the initiator tRNA in eukaryotes. met Met-tRNAe participates in translation elongation. Many eukaryotic initiation factors ( eIFs ) are required in eukaryotic initiation. Cap and poly A tail participate in the initiation. met Met-tRNAi binds to 40S subunit before mRNA binding Homework Read eukaryote translation initiation on P.377 Draw a comparison between prokaryotic and eukaryotic translation initiation. C Translation Elongation ( in prokaryotes ) Elongation is a process of repeated ribosomal cycles of amino acid addition. With the formation of the 70S initiation complex elongation begins. The ribosomal cycle can be divided into three steps, entrance ( registration ), peptide bond formation and translocation. When one ribosomal cycle is completed, the nascent peptide is an amino acid residue longer. There is another extended sense of ribosomal cycle, which is the cycle of translation. It also has three stages, initiation, elongation and termination. When such a ribosomal cycle is completed, one polypeptide is synthesized. The Process of Elongation Cycle 1. Entrance Entrance begins when the P site is occupied by the initiator tRNA. The complex of an elongation factor Tu and GTP ( EFTu-GTP ) is required to deliver the aminoacyl-tRNA to the A site according to the codon-anticodon base-pairing The energy is consumed in this step by hydrolysis of GTP catalyzed by EFTu. The EFTu-GDP is released. Another elongation factor EFTs interacts with EFTu and displaces GDP. EFTu-GDP + EFTs-----EFTuTs + GDP The EFTu-GTP complex is regenerated when GTP displaces EFTs. EFTuTs + GTP-------------EFTu-GTP + EFTs Thus EFTu has GTPase activity and EFTs is a GTP/GDP exchange protein. 2. Peptide Bond Formation After aminoacyl-tRNA delivery the A and P sites are both occupied. The two amino acids ( fMet and AA ) are linked by forming a peptide bond. The 23SrRNA ( in eukaryotic ribosome the 28SrRNA ) catalyzes the peptide bond formation between the acyl group of the fMet residue and the alpha amino group of the next AA residue. So, protein biosynthesis is directional, from N-terminal to C-terminal. 3. Translocation A complex of elongation factor G and GTP ( EFG-GTP ) binds to the ribosome and in an energy-consuming step. The discharged tRNA from P site goes to E site and then exits. The peptidyl-tRNA is moved from the A site to P site and the mRNA moved by one codon relative to the ribosome. EFG and GDP are released, the former being reused. A new codon is now present in the vacant A site. The elongation cycle is repeated until one of the stop codons ( UAA, UAG,UGA ) appears in the A site. D . Termination Termination is the process of release of the newly synthesized polypeptide. Protein factors called release factors interact with stop codons and cause release of the newly synthesized polypeptide. There are three RFs in prokaryotes. RF1 recognizes UAA and UAG and RF2 recognizes UAA and UGA. RF3 helps either RF1 or RF2 to carry out the reaction. The release factors make 23SrRNA transfer the polypeptide to water. This reaction is driven by GTP and RF3 has GTPase activity. Thus the polypeptide is released from the ribosome. IF1 binds to the 70S ribosome. The two subunits of the 70S ribosome then dissociate. They participate in a new round of translation initiation. There is a single release factor in eukaryotes the eRF. It performs the roles carried out by RF1, RF2 and RF3 in prokaryotes. When a ribosome has begun translating an mRNA molecule and has moved about 70 - 80 nt from the initiation codon, a second ribosome can assemble on the mRNA and start translation. When this second ribosome has moved along a third can begin and so on. Multiple ribosomes on a single mRNA are called polysomes. In this way protein synthesis performs with high efficiency. Section Three Protein Folding and Posttranslational Processing The newly synthesized polypeptide must undergo folding and posttranslational processing so that it becomes the functional protein with natural conformation. A Posttranslational Processing of Newly Synthesized Polypeptide 1.N-terminal processing includes removing N-formyl group, or N-fMet, ( in prokaryotes ) or N-Met ( in eukaryotes ), or several amino acid residues at the N-terminal ( in both prokaryotes and eukaryotes ). In eukaryotes N-terminal signal sequence ( signal peptide ) of membrane or secretory proteins are removed. There may be acetylation of N-terminal amino acid residue. C-terminal processing may also occurs. 2. Proteolytic Processing and Protein Splicing Some proteins may undergo proteolytic processing. The well-known example is the proteolytic processing of polyprotein POMC. POMC is cleaved into different peptide hormones in different tissures. It has been found that certain proteins in prokaryotes have protein ‘ intron’. The protein ‘ intron’ catalyzes self-splicing. 3. There are a lot of amino acid residue modification patterns : a. methylation b. acetylation c. phosphorylation d. glycosylation e. hydroxylation f. disulfide bond formation g. farnesylation h. covalent binding of the prosthetic group and so on B Protein Folding As each nascent polypeptide emerges from the ribosome, it begins to fold into its final three-dimensional structure. 1. Two Models of Protein Folding: a. hierarchical folding model secondary structuresuper-secondary structure tertiary structure b. molten globule model hydrophobic interactionmolten globule 2. Molecular Chaperones Participate in Protein Folding Molecular chaperones are a kind of proteins which help proteins to fold. They have following functions a. binding to the hydrophobic areas of the unfolded protein thus preventing them from abnormal aggregation b . establishing an isolated environment for protein folding c. promoting protein folding and de-polymerization d. in case of stress, unfolding the folded protein Molecular chaperones are divided into two groups. a. molecular chaperones which bind to ribosome such as TF, NAC. b. molecular chaperones which do not bind to ribosome such as Hsp families,PDI,and PPI. Molecular chaperones’ work is driven by ATP. Homework Read the text on pp.383, 384,and 385. Summary the contents on these pages in several sentences. for example Hsp70 1.The structure and function of Hsp70 2. Two helpers of Hsp70 3. The function of Hsp40 4. The function of GrpE Tell the story of E coli GroEL in five sentences. C Polymerization of Subunits, Multi-subunit Protein Formation HbA formation Free alpha peptide binds to nascent beta peptide and alpha-beta dimer forms. The dimer binds to two heme molecules. Two heme-containing alpha-beta dimers interact each other and form HbA. Section Four Clinical Relatives in Protein Synthesis Protein synthesis is a regulated process. The protein synthesis machinery responds the environmental stimulation. The synthesis of ferritin, a ferrous ion (Fe2+) binding protein, is regulated by Fe2+. Fe2+ binds to the 5’UTR of the ferritin mRNA and stimulates ferritin synthesis. A Many viruses co-opt the host cell protein synthesis machinery. 1.Virus mRNA is more efficiently translated than host cell mRNA. 2. Viruses make abundant mRNA. 3.Some viruses can inhibit host cell mRNA binding to 40S subunit. B Many antibiotics work because they selectively inhibit protein synthesis in bacteria. Chloramphenicol inhibits prokaryotic peptidyl transferase. Streptomycin binding to 30S subunit causes mRNA misreading. Tetracycline binding to 30S subunit interferes with aminoacyl-tRNA binding. tyr Puromycin is an analog of Tyr-tRNA and can enter the A site during protein synthesis. If puromycin occupies A site, translation elongation stops. Puromycin was used in the study on protein synthesis. Cycloheximide inhibits eukaryotic peptidyl transferase. It is used in studies on protein metabolism. Some bioactive substances that can inhibit cell or virus protein synthesis diphtheria toxin Diphtheria toxin is a kind of enzyme. It transfers ADP-ribose group from NAD+ to the eukaryotic elongation factor 2 (EF2). That results in inactivation of EF2 and translation stops. interferon When mammalian cell is infected by virus, the host cell can synthesize interferon. Interferon has two ways to combat virus. 1 It induces synthesis of HCI, a kind of protein kinase. HCI catalyzes phosphorylation of eIF2. The phosphorylated eIF2 is inactive. Protein synthesis stops 2 Interferon induces an enzyme for the synthesis of 2’5’oligoA. 2’5’oligoA is synthesized in the cell. 2’5’oligoA activates RNase L RNase L hydrolyzes virus mRNA.