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Chapter 12. Protein biosynthesis Protein biosynthesis is a process to express genetic information in living cells, which is called translation. The genetic information flows as: Transcription DNA Reverse transcription Translation RNA Protein 1. Components of Protein Biosynthesis Protein biosynthesis requires: amino acids, mRNA, tRNA, ribosomes, protein factors, and synthetic enzymes. 1) Messenger RNA: a template for protein biosynthesis, which is read in a 5’3’ direction. Each three nucleotides form a codon representing for a specific amino acid. Thus, the base sequence of an mRNA molecule determines the amino acid sequence of the protein. Codons in mRNA U C A G U C A G Phe Phe Leu Leu Ser Ser Ser Ser Tyr Tyr Stop Stop Cys Cys Stop Trp U C A G Leu Leu Leu Leu Pro Pro Pro Pro His His Gln Gln Arg Arg Arg Arg U C A G Ile Ile Ile Met Thr Thr Thr Thr Asn Asn Lys Lys Ser Ser Arg Arg U C A G Val Val Val Val Ala Ala Ala Ala Asp Asp Glu Glu Gly Gly Gly Gly U C A G mRNA in eukaryotes is usually monocistronic: one mRNA encodes only a single polypeptide chain. mRNA in prokaryotes usually encodes more than one polypeptide chain. This is called polycistronic. A) Degeneracy of codons: refers to the fact that an amino acid has more than one codon. one of the consequences of degeneracy is that a mutation which produces a base change in DNA may not result in an amino acid change in the encoded protein. Synonyms: refers to the codons for the same amino acid. e.g. GUU, GUC, GUA, GUG represent for Val. B) Universility of codons: this genetic code system is used by all living organisms except in some cases: in cytosol in mitochondria AUA UGA AGA CGG CUN Ile Stop Arg Arg Leu Met Trp Stop (animal) Trp (plant) Thr (yeast) C) Reading frames: refer to the different combinations for each three nucleotides that are read as a codon: each mRNA sequence can be read in three possible reading frames. Reading frame 1: UUA UGA GCG CUA AAU Leu Stop Ala Leu Asn Reading frame 2: U UAU GAG CGC UAA AU Tyr Glu Arg Stop Reading frame 3: UU AUG AGC GCU AAA U Met Ser Ala Lys D) Open reading frames: refer to the runs of codons that start with ATG and end with TGA, TAA, or TAG. The open reading frames can be used to predict the protein sequence encoded. 2) Transfer RNAs: the fidelity of protein biosynthesis requires tRNAs to serve as adapters that can recognize the correspondent codons and carry amino acids to the right positions in translation. Each tRNA only brings with it an amino acid, and recognizes and binds to a specific codon. Secondary structure of tRNA 3 A A C A C A 5 A A A A A A A A A A A TC loop U C A A A A A A A A A G A T C 鴠 A G A A A A G extra arm A DHU loop U A Anticodon loop Tertiary structure of tRNA Codon-anticodon interaction by base pairing mRNA 5’ CUA GAU 3’ tRNA 3’ 5’ Wobble base pairing: base pairing between the 3’ position of the codon and 5’ position of the anticodon may occur by a nonstandard way. This allows one tRNA to recognize more than one codon. Examples of wobble base pairing Anticodon wobble position base C A G U Codon wobble position base G I U C A C U G U A Wobble base pairing of inosine with three nucleosides rib o se rib o se rib o se N N O N H N O N N O O N N c ytid ine Ino sine O N N N rib o se rib o se N N N H H N N N H H H Ino sine N H H N N N O N rib o se urid ine Ino sine a d e no sine 3) rRNAs and ribosomes: As the site of protein biosynthesis, ribosome is made up of two subunits, one is large and another is small. Composition of ribosomes in eukaryotes and prokaryotes Subunit size Eukaryotic ribosome (80S) Prokaryotic ribosome (70S) large subunit small subunit large subunit small subunit 60S 40S 50S 30S rRNAs 5S, 5.8S, 28S 18S 5S, 23S 16S Proteins 49 33 35 21 A) Polysomes: several ribosomes bind to and translate a single mRNA molecule simultaneously B) Free ribosomes: ribosomes occur free in the cytosol, usually synthesizing proteins of cytosol, nucleus, mitochondria or other organelles C) Membrane bound ribosomes: ribosomes bind to the membrane of rough endoplasmic reticulum, usually synthesizing secretory proteins or membrane proteins. Polysomes 4) Aminoacyl-tRNA synthetase: is also called amino acid activating enzyme, which catalyzes the following reactions. Synthetase tRNA + Met CH3 CH3 S S CH2 CH2 CH2 transformylase O H2N CH C O Met (Met -tRNAi tRNAf Met O HC CH2 O HN CH C O tRNAf ) Met 10 N -formyl tetrahydrofolate (fMet-tRNAi tetrahydrofolate ) Met 2. Steps of Protein Biosynthesis The steps of protein biosynthesis include: initiation, elongation, and termination or release. 1) Initiation: Translation begins with the assembly of an initiation complex consisting of an mRNA, a ribosome, and the initiator tRNA (fMet-tRNAiMet or MettRNAiMet ) . The process requires a number of protein factors, known as initiation factors. Formation of the initiation complex in eukaryotic translation mRNA Cap Cap AUG AUG Met Met eIF1,2,3,4, GTP 60S + eIF5 Met Cap Cap UAC AUG AUG UAC 40S ATP ADP+Pi eIF1,2,3,4,5, GDP+Pi Cap AUG UAC Met-tRNA Initiation complex In prokaryotes, initiation factors IF1 and IF3 bind to the 30S subunit while IF2 binds to GTP·fMet-tRNAiMet. The two complexes and mRNA combine to form a pre-initiation complex, releasing IF3. The 50S subunit binds with this complex, with hydrolyzation of the bound GTP to GDP and Pi, and release of IF1 and IF2, to form a completed initiation complex. Formation of the initiation complex in prokaryotic translation IF2•GTP·fMet-tRNAiMet 30S IF1, IF3 IF1 30S IF3 IF1 30S mRNA mRNA IF3 IF2•GTP·fMet-tRNAiMet 50S mRNA IF1, IF2, GDP fMet Prokaryotic and eukaryotic initiation factors Prokaryotic eukaryotic IF1 eIF 1 IF2 eIF2a eIF 2b eIF 2c IF3 eIF3 eIF4a eIF4b eIF4c eIF4d eIF4e eIF4f eIF5 eIF6 Function IF1 binds to small subunit before mRNA bi nding. eIF1 assists mRNA binding. Bind initiator tRNA, stabilize ternary complex, cause GTP/GDP exchange. Bind to the small subunit, assist mRNA binding, cause dissociation of subunits after translation. Recognize and bind the mRNA cap, assist mRNA binding, hydrolyze ATP to drive scanning for the initiator codon. Promotes GTP hydrolysis and release of other initiator factors. Assists subunit dissociation. 2) Elongation: Elongation of polypeptide chain consists of a series of cycles, called ribosomal cycles, each of which forms a new peptide bond. Three steps: entry, peptide bond formation, and translocation. A) Entry of aminoacyl-tRNA to the A site of ribosome (A. in prokaryotes, B. in eukaryotes. AA = aminoacyl) A. Ts T u -Ts + G T P A A -tR N A T u -G T P GDP A A -tR N A -T u -G T P mRNA Ts Pi T u -G D P + A A -tR N A -m R N A B. e E F -1 + G T P e E F -1 A A -tR N A e E F -1 -G T P GDP A A -tR N A -e E F -1 -G T P mRNA e E F -1 Pi e E F -1 -G D P + A A -tR N A -m R N A B) Peptide bond formation dipeptidyl-tRNA P site A site P site N H2 CH R1 H2N R2 CH CO H2N R1 CO Peptidyltransferase NH CH CH O CO OH CO tRN A 1 O tRN A 1 O tRN A 2 A site R2 tRN A 2 C) Translocation: Translocation is a process involves the shift of the newly formed peptidyl(n+1)-tRNA from the A site to the P site, with release of the deacylated tRNA from the ribosome. This process is mediated by another elongation factor, EF-G in prokaryotes, or eEF2 in eukaryotes. The translocation step in protein biosynthesis Peptidyl-tRNA Peptidyl-tRNA tRNA P 5’ Translocation A P 5’ 3’ GTP-EFG EFG + GDP + Pi A 3’ D) Termination: when a ribosome moves onto the stop codon of mRNA, the stop codon in the A site cannot be recognized by any aminoacyl-tRNA molecules. Instead, release factors interact with the mRNA-ribosome complex, leading to discharge of the newly synthesized polypeptide from the complex. Termination of protein biosynthesis NH 3+ Peptidyl-tRNA NH3+ Peptidyl-tRNA eRF-GTP A P UAG 5’ 5’ UAG A eRF-GTP P 3’ 3’ Pi + H 3N Peptide chain 3’ P 5’ 40S 60S tRNA eRF-GDP eIF6 eRF-GDP 5’ tRNA mRNA UAG A 3’ Elongation and termination factors in prokaryotes and eukaryotes Prokaryotic eukaryotic Function Elongation factors: EFTu eEF1 EFTs eEF1 EFG eEF2 Termination factors: RF1 eRF RF2 RF3 Bind amino acyl tRNA.GTP Assist in the exchange of GTP and GDP Hydrolyze GTP, translocate mRNA along ribosome RF1 recognizes UAA, UAG. eRF recognizes all three termination codons, and binds and hydrolyzes GTP, causing release of peptide and tRNA from the ribosome. Recognizes UAA, UGA. Binds GTP and interacts with RF1 and RF2. (Eukaryotic) 3. Posttranslational Processing Newly synthesized polypeptides usually undergo structural changes called posttranslational processing. The most important posttranslational processing: modification and folding. 1) Posttranslational modification: A) Modification of protein primary structures Removal of the N-terminal Met residue H2 O O O O HC NH CH C NH CH C CH2 ¯ HCOO O AA3 AAn + COO NH 3 Deformylase R CH CH2 CH2 CH2 SCH3 SCH 3 Met-specific aminopeptidase O + H2 O Methionine NH3 CH C R AA3 AAn COO C O NH CH R C AA 3 AAn COO Posttranslational processing of human preproinsulin + N H 3+ COO H2O H 3N S Signal peptide S S S COO - - Signal peptidase Proinsulin Preproinsulin 6H2O 4AA + Peptide C B chain A chain + + H 3N H 3N S COO S S S Insulin COO - - B) Glycosylation: occurs in most membrane and secretary proteins, such as glycoproteins. Two types of glycoproteins in humans: O-linked and N-linked. Formed in endoplasmic reticulum and Golgi apparatus. N-linked O-linked CH 2 OH CH 2 OH O NH NH OH CO CH 2 CH HO O NH OH CO H O NH NH CO CO CH 3 CH 3 O CH 2 CH CO C) Modification of protein on higher-level structures Acetylation of the amino terminus: Acetyl-SCoA + H2N-protein Acetyl-NH-protein + HSCoA Phosphorylation: ATP ADP Protein kinase Protein Pi Phosphoprotein Phosphatase H2O 2) Folding of newly synthesized polypeptides Newly synthesized polypeptide chains usually undergo folding, a process that requires protein factors called molecular chaperones. Two types of molecular chaperones: chaperones and chaperonins. The major function of molecular chaperones is to assist the correct folding of nascent polypeptide chains by blocking their hopeless entangling or insignificant intermolecular interactions. Molecular chaperones belong to the “heatshock protein (HSP) family”. A) Chaperone proteins: include HSP70, HSP40, and GrpE. The binding-release cycle of chaperone proteins with a nascent polypeptide earns time for the proper folding of the unfolded polypeptide chain. The cycle continues until the polypeptide chain is folded to a native conformation. The binding-release cycle of a chaperon-polypeptide complex Pi ATP HSP40-Polypeptide-HSP70-ADP GrpE ADP HSP70-ATP HSP40-Polypeptide HSP40-Polypeptide-HSP70-ATP HSP40 Polypeptide Polypeptide + HSP70-ATP B) Chaperonins: are also heat-shock proteins. They participate in the folding of a variety of proteins by forming a cylindrical structure (a ring) enclosing a central cavity. The target polypeptide chain enters the central cavity of the folding machine, where it is properly folded and is then released. The entering-folding process repeats until a native 3D structure of the protein is formed. A folding cycle of a polypeptide by GroEL-GroES chaperonins in E. coli cell GroES Folded polypeptide polypeptide + GroEL GroEL-polypeptide PolypeptideGroEL-GroES complex GroEL GroES 4. Protein targeting Protein targeting is a process in which a newly synthesized protein is delivered to a specific extracellular or intracellular location. Secretory proteins are first synthesized by ribosomes bound to the rough ER (RER), with a signal sequence (or called signal peptide) at the N-terminal end, which directs the protein to be delivered to its functioning place. A. Hydrophobic area Signal peptidase cleavage site NSecretory protein Signal peptide Internal signal peptide B. N- Signal peptide Stop-transfer sequence Type III integral membrane protein (A) Signal peptides of secretory proteins. (B) Type III integral membrane proteins with signal-peptide, internal signal-peptide, and stop-transfer sequences. The signal peptide directs a newly synthesized secretary protein to enter into the RER lumen SRP RER lumen SRP receptor signal peptidase Signal peptide RER membrane SRP: signal recognition particle 5. Clinical correlation of protein biosynthesis Protein biosynthesis is the means to express the genes that control metabolisms in cells. Any mistake occurs in protein biosynthesis may result in severe consequences in metabolism. 1) Molecular Diseases: refer to those resulting from abnormal protein structures due to mutation of genes. Sickle-cell anemia: a result from the replacement of an amino acid residue at position 6 of the chain, glutamate , by another one, valine. Position of -chain Hemoglobin A Hemoglobin S 1 2 3 4 5 6 7 8 Val-His-Leu-Thr-Pro-Glu-Glu-LysVal-His-Leu-Thr-Pro-Val-Glu-Lys- 2) Action of some antibiotics: they carry out antimicrobial activities via inhibition of protein synthesis in the microorganism, such as tetracyclines, streptomycin, chloramphenicol, and so on. Some antibiotic inhibitors of protein biosynthesis Antibiotic Tetracycline Streptomycin Chloramphenicol Erythromycin (prokaryotes) Puromycin Cycloheximide Action Steps affected Binds to the 30S subunit, inhibits binding of aminoacyl-tRNA Binds to the 30S subunit, inhibits binding of fMet-tRNA and causes misreading of mRNA Binds to the 50S subunit, inhibits peptidyltransferase activity Binds to the 50S subunit entry of aminoacyl-tRNA (prokaryotes) initiation, elongation (prokaryotes) Acts as an analog of aminoacyltRNA and binds to the A site Blocks peptidyltransferase activity Elongation (prokaryotes & eukaryotes) Elongation (eukaryotes) Elongation (prokaryotes) Translocation 3) Effect of some biological molecules : Interferons (IFNs) are cytokines produced during immune response to antigens, especially to viral infections. Two functions of IFNs: cause viral RNA degradation and inhibit protein biosynthesis in cells. IFN protein kinase phosphorylation of eIF-2a inhibition of protein biosynthesis in cells inhibition of the viral replication.