BCM II: Protein Synthesis PDF

BIOCHEMISTRY LC 7: PROTEIN SYNTHESIS DR. BRENDO V. JANDOC | 02/11/2025 -...

BIOCHEMISTRY LC 7: PROTEIN SYNTHESIS DR. BRENDO V. JANDOC | 02/11/2025 - ○​ any alteration in the nucleic acid sequence COURSE OUTLINE → improper amino acid inserted into the I.​ INTRODUCTION II.​ RIBOSOMES AND PROTEIN ASSEMBLY polypeptide chain → disease or death of the A.​ Structure and components of ribosomes organism B.​ From gene to protein C.​ Nucleolus and ribosomes ​ Translation D.​ Medical relevance ○​ process by which ribosomes convert the III.​ GENETIC CODE information carried by mRNA to the A.​ Codons synthesis of new proteins B.​ Characteristics of genetic code ○​ requires the cleavage of “high-energy” C.​ Colinearity of gene and product D.​ Overlapping genes phosphoanhydride bonds endergonic IV.​ COMPONENTS REQUIRED FOR TRANSLATION A.​ Amino acids B.​ tRNA C.​ mRNA D.​ Aminoacyl-tRNA synthetases E.​ Functionally competent ribosomes F.​ Protein factors G.​ ATP and GTP as energy sources V.​ AMINO ACID ACTIVATION: FORMATION OF AMINOACYL-TRNAS A.​ Adaptor molecules B.​ Aminoacyl-tRNA synthetases VI.​ CODON RECOGNITION BY TRNA (CODON-ANTICODON RECOGNITION) A.​ Antiparallel binding between codon and anticodon B.​ Wobble hypothesis C.​ Rules governing base pairing of the codons 3rd base position VII.​ PROTEIN SYNTHESIS IN PROKARYOTES A.​ Initiation B.​ Elongation C.​ Termination D.​ Energy requirements for protein Figure 1. Protein synthesis or translation. tRNA = transfer RNA; rRNA = ribosomal RNA; mRNA = messenger RNA; UTR = synthesis untranslated region. E.​ Role of high energy requirements during translation F.​ Polysomes II. RIBOSOMES AND PROTEIN ASSEMBLY VIII.​ PROTEIN SYNTHESIS IN EUKARYOTES ​ Ribosomes A.​ Initiation ○​ large ribonucleoprotein particles B.​ Elongation C.​ Termination ○​ coordinate the interaction of mRNA and D.​ Gene expression regulation tRNAs during protein synthesis IX.​ PROTEIN LOCALIZATION IN EUKARYOTES ○​ products of individual genes (ribosomal A.​ Protein synthesis on genes) membrane-bound ribosomes ​ Proteome B.​ Protein targeting ○​ All proteins produced by a cell at any given X.​ POSTTRANSLATIONAL MODIFICATION OF time POLYPEPTIDE CHAINS A.​ In endoplasmic reticulum B.​ Beyond the endoplasmic reticulum STRUCTURE AND COMPONENTS OF C.​ Protein degradation RIBOSOMES XI.​ DRUGS AND INHIBITORS OF PROTEIN ​ Ribosomes – made of small and a large subunit SYNTHESIS IN EUKARYOTES A.​ Therapeutic drugs B.​ Inhibitors PROKARYOTIC RIBOSOMES C.​ Toxins ​ a bacterial cell contains about 20000 ribosomes XII.​ SUMMARY (about 25% of its mass) XIII.​ REFERENCES ​ Contains – three different types of of rRNA molecules; up to 83 proteins I. INTRODUCTION ​ Escherichia coli ribosome ​ Genetic information ○​ sedimentation coefficient of 70 S ○​ stored in the chromosomes ○​ Approximately: 65% RNA & 35% protein ○​ transmitted to daughter cells through DNA ​ Sedimentation coefficient replication ○​ measure of the rate of sedimentation in an ○​ expressed through transcription to RNA ultracentrifuge of a molecule suspended in a ○​ translation into polypeptide chains less dense solvent ○​ measured in Svedberg units (S) ○​ S values are not additive BATCH TANNAWAG 1E​ 1 BIOCHEM LC 7: PROTEIN SYNTHESIS ​ Dr. JANDOC, B. 02/11/2025 ​ Dalton – unit of atomic or molecular mass MITOCHONDRIA AND CHLOROPLASTS ​ Prokaryotic 70S Ribosome ​ have their own ribosomes ○​ molecular weight of 2.5 million daltons (MDa) ​ resemble those of prokaryotes ○​ can be dissociated into: ○​ Structure ○​ 30 S ○​ sensitivity to antibiotic inhibitors of translation ​ smaller subunit much large ​ contains – large 16 S rRNA and 21 proteins FROM GENE TO PROTEIN ​ site where genetic information is decode ​ Cell Nucleus ​ has a proofreading mechanism ○​ directs the production of endogenous proteins ○​ 50 S (protein synthesis) ​ larger subunit ​ Nuclear RNA ​ consists of smaller rRNAs (5 S) with 120 ○​ bound to nuclear RNA-binding proteins for ribonucleotides, larger rRNAs (23 S) with stabilization ~2900 ribonucleotides, and 3-35 proteins ​ Mature RNA ​ ​ provides peptidyltransferase activity ○​ released from the nucleus into the cytoplasm ○​ associates with ribosomes Figure 2. Structure & Components of Prokaryotic Ribosomes EUKARYOTIC RIBOSOMES Figure 4. From gene to protein ​ much larger (80 S, 4.2 MDa) ​ consists of NUCLEOLUS AND RIBOSOMES ○​ 40 S subunit ​ Nucleolus ​ Contains – 18 S rRNAs (1900 bases) & 33 ○​ morphologically and functionally specific region proteins in the cell nucleus ○​ 60 S subunit ○​ where ribosomes are synthesized ​ Contains – 5 S rRNAs (120 bases), 5.8 S ○​ In humans: rRNAs (160 bases), 28 S rRNAs (4800 ​ rRNA genes (200 copies per haploid bases) & 50 proteins genome) → transcribed by RNA polymerase I → formation of 45 S rRNA precursors → packaged with ribosomal proteins (from the cytoplasm) → cleavage → three of the four rRNA subunits → transfer from the nucleus and released into cytoplasm with the separately synthesized 5S → subunits formation of functional ribosomes ​ Small RNAs ○​ Small nuclear RNAs (snRNA) ​ family of RNA molecules that bind specifically with small number of nuclear ribonucleoprotein particles (snRNP, pronounced “snurps”) ​ important roles in the post-transcriptional modification of RNA molecules ​ base-pair with pre-mRNA and with each Figure 3. Structure & Components of Eukaryotic Ribosomes other during RNA splicing ​ Made of non-coding RNA and participates in splicing ​ ​ ​ BATCH TANNAWAG 1E ​ 2 BIOCHEM LC 7: PROTEIN SYNTHESIS ​ Dr. JANDOC, B. 02/11/2025 ​ Small nucleolar RNAs (snoRNA) ○​ assist in processing of pre-rRNAs and in the assembly of ribosomes Figure 6. Use of the genetic code table to translate the codon AUG. A = adenine; G = guanine; C = cytosine; U = uracil. The abbreviations for many common amino acids are shown as examples. ​ ​ ​ ​ ​ TERMINATION (STOP OR NONSENSE) CODONS ​ Three of the codons, UAA, UAG, and UGA, do not code for amino acids but, rather, are termination codons. When one of these codons Figure 5. Nucleolus and synthesis of ribosomes appears in an mRNA sequence, synthesis of the polypeptide coded for by that mRNA stops. MEDICAL RELEVANCE ​ a variety of chemical compounds (naturally as poisons or synthetic products) are used for cancer therapy by inhibition of transcription or translation ​ Produce effectors including growth factors which are proteins which targets the process of transcription and even translation to treat cancers. III. GENETIC CODE ​ System of RNA sequences that designate particular amino acids in the process of translation Figure 7. Genetic code for all amino acids in mRNA CODONS ​ genetic words ​ composed of 3 nucleotide bases ​ usually presented in the mRNA language of ○​ adenine (A) ○​ guanine (G) ○​ cytosine (C) ○​ uracil (U) ​ nucleotide sequences are written from the 5’-end to the 3’-end ​ 4 nucleotide bases Figure 8. Abbreviated code ○​ used to produce the 3-base codons → 64 different combinations CONSEQUENCES OF ALTERING THE NUCLEOTIDE SEQUENCE HOW TO TRANSLATE A CODON ​ changing a single nucleotide base on the mRNA ​ This table (or “dictionary”) can be used to chain (point mutation) → 1 of the following translate any codon and, thus, to determine which results amino acids are coded for by an mRNA ○​ Silent mutation – the new codon may code for sequence. For example, the codon 5'-AUG-3' the same amino acid codes for methionine ​ (see Figure 6) ​ Ex: if the serine (Ser) codon UCA is given a ○​ AUG is the initiation (start) codon for different third base, U, to become UCU, it translation. still codes for Ser. This is termed a “silent” ​ Sixty-one of the 64 codons code for the 20 mutation. common amino acids. ○​ Missense mutation – the new codon may code for a different amino acid ​ Ex: If the Ser codon UCA is given a different first base, C, to become CCA, it will code for a different amino acid (in this case, proline). The substitution of an incorrect amino acid is called a “missense” mutation. BATCH TANNAWAG 1E ​ 3 BIOCHEM LC 7: PROTEIN SYNTHESIS ​ Dr. JANDOC, B. 02/11/2025 ○​ Nonsense mutation – the new codon may NEAR UNIVERSALITY become a termination codon ​ the specificity of the genetic code has been ​ Ex: If the Ser codon UCA is given a different conserved from very early stages of evolution second base, A, to become UAA, the new ​ only slight differences in the manner the code is codon causes termination of translation at translated that point and the production of a shortened ​ exceptions in the universality of the genetic code (truncated) protein. The creation of a are found in human mitochondria termination (stop) codon at an inappropriate ○​ UGA – designates tryptophan place is called a “nonsense” mutation. ○​ AUA – codes for methionine ​ ○​ additional stop codons – AGA & AGG ○​ Other mutations – can alter the amount or ​ Ex: UUU – code for phenylalanine structure of the protein produced by translation ​ Trinucleotide repeat expansion ​ Splice site mutations ​ Frame-shift mutations Figure 10. Deviations from the universal genetic code REDUNDANT/DEGENERATE CODE ​ more than 1 codon can specify a single amino acid ​ all amino acids, except methionine and tryptophan, have more than one codon ○​ Synonyms ​ codons that designate the same amino acid ​ synonymous codons usually differ only in the 3rd base of the codon ○​ Amino Acids Having More Than One Codon Figure 9. Possible effects of changing a single nucleotide base in ​ first 2 bases in the codon are usually the the coding region of a messenger RNA chain. A = adenine; C = same cytosine; U = uracil. ​ base in the third position often varies ​ For example, arginine (Arg) is specified by six NONRANDOM ARRANGEMENT OF CODONS different codons (see Figure 6). Only Met and Trp ​ Nonrandom evolution of genetic code → have just one coding triplet. minimizes the deleterious effects of point mutations UNAMBIGUOUS CODONS ​ Most Synonyms ​ Each codon specifies no more than one amino ○​ share the first two acid ○​ Ex: UUU & UUC = Phenylalanine ​ Codons with Different Nucleotides in the 1st NONOVERLAPPING AND COMMALESS Position ○​ tend to specify chemically similar amino acids ​ the code is read from a fixed starting point as a ○​ Ex: Valine & Leucine continuous sequence of bases, taken 3 at a time, ​ Codons with 2nd Position Pyrimidines and without spacer bases ○​ encode mostly hydrophobic amino acids ​ if 1 or 2 nucleotides are deleted or inserted to the ​ interior of a message sequence → frameshift ○​ Even though there is a mutation, if valine is mutation reading frame is altered replaced by leucine, since both are ​ if 3 nucleotides are added → new amino acid is hydrophobic, both are buried inside the protein added to the polypeptide (reading frame is not core. Still the structure is functional. affected) ​ Codons with 2nd Position Purines ​ if 3 nucleotides are deleted → an amino acid is ○​ encode mostly polar/hydrophilic amino acids lost (reading frame is not affected) ​ example, AGCUGGAUACAU is read as AGC CHARACTERISTICS OF THE GENETIC CODE UGG AUA CAU ​ Usage of the genetic code is remarkably consistent throughout all living organisms. It is assumed that once the standard genetic code evolved in primitive organisms, any mutation that altered its meaning would have caused the alteration of most, if not all, protein sequences, resulting in lethality. SPECIFICITY ​ Specific (unambiguous), because a specific codon always codes for the same amino acid Figure 11. Frame-shift mutations as a result of addition or deletion of a base can cause an alteration in the reading frame of mRNA. BATCH TANNAWAG 1E ​ 4 BIOCHEM LC 7: PROTEIN SYNTHESIS ​ Dr. JANDOC, B. 02/11/2025 COLINEARITY OF GENE AND PRODUCT ​ only 20 amino acids → some amino acids have ​ The product of the gene is the peptide specified more than 1 specific tRNA molecule by the sequence of the expressed regions of the ○​ called adaptor molecules genes ​ carry a specific amino acid ​ recognize the codon for that amino acid OVERLAPPING GENES ​ some viruses code for more proteins than 1.​ Amino Acid Attachment Site would be predicted from their nucleotide content ○​ at the 3’-end of the tRNA ○​ some of the expressed portions of the viral ○​ carboxyl group of the amino acid is in an ester genome overlap and code for multiple linkage with the 3’-hydroxyl of the ribose products in different reading frames. moiety of the adenosine nucleotide in the -CCA sequence at the 3’-end of the tRNA ○​ when a tRNA has a covalently attached amino acid → said to be “charged” ○​ when a tRNA is not bound to an amino acid → said to be “uncharged” ○​ the amino acid that is attached to a tRNA → said to be “activated” Figure 12. Reading frame of mRNA. A. For any given mRNA sequence, there are three possible reading frames (1, 2, and 3). B. An AUG near the 5’-end of the mRNA (the start codon) sets the reading frame for translation of a protein from the mRNA. The codons are read in linear order, starting with this AUG. (The other potential reading frames are not used. They would give proteins with different amino acid sequences.) IV. COMPONENTS REQUIRED FOR TRANSLATION ​ A large number of components are required for the synthesis of a protein. These include all the amino acids that are found in the finished product, the mRNA to be translated, transfer RNA (tRNA) for each of the amino acids, functional Figure 14. Amino acid Activation ribosomes, energy sources, and enzymes as well as non catalytic protein factors needed for the 2.​ Anticodon initiation, elongation, and termination steps of ○​ 3-base nucleotide sequence of the tRNA polypeptide chain synthesis. molecule ○​ recognizes a specific codon on the mRNA AMINO ACIDS which specifies the insertion into the growing ​ all amino acids must be present peptide chain of the amino acid carried by the ​ amino acid deficiency or absence → tRNA translation stops at the codon specifying that amino acid mRNA ​ template for the synthesis of the desired polypeptide chain AMINOACYL-TRNA SYNTHETASES ​ This family of enzymes is required for attachment of amino acids to their corresponding tRNAs. ​ Each member of this family recognizes a specific amino acid and all the tRNAs that correspond to that amino acid ​ Aminoacyl-tRNA synthetases catalyze a two-step reaction that results in the covalent attachment of the carboxyl group of an amino acid to the 3'-end of its corresponding tRNA. ​ The overall reaction requires adenosine triphosphate, which is cleaved to adenosine monophosphate (AMP) and inorganic Figure 13. Complementary, antiparallel binding of the anticodon for methionyl-tRNA (CAU) to the mRNA codon for methionine pyrophosphate (PPi) (AUG), the initiation codon for translation. ​ The extreme specificity of the synthetases in recognizing both the amino acid and its cognate tRNA tRNA contributes to the high fidelity of translation ​ specific type of tRNA is required per amino of the genetic message. acid ​ Their synthetic activity, the aminoacyl-tRNA ○​ humans – at least 50 species of tRNA synthetases have a “proofreading” or “editing” ○​ bacteria – 30-40 species activity that can remove an incorrect amino acid from the enzyme or the tRNA molecule. BATCH TANNAWAG 1E ​ 5 BIOCHEM LC 7: PROTEIN SYNTHESIS ​ Dr. JANDOC, B. 02/11/2025 FUNCTIONALLY COMPETENT RIBOSOMES ii.​ Rough Endoplasmic Reticulum-Associated ​ Ribosomes Ribosomes ○​ large complexes of: protein & rRNA ○​ responsible for synthesizing proteins that ○​ consist of 2 subunits: large & small are to be: ​ exported from the cell PROKARYOTIC AND EUKARYOTIC ​ integrated into: RIBOSOMES - plasma membrane ​ similar in structure - endoplasmic reticulum membrane ​ serve the same function (factories for protein - Golgi membrane synthesis) ​ incorporated into lysosomes ii. Mitochondria RIBOSOMAL SUBUNIT ○​ have their own set of ribosomes ​ Large Ribosomal Subunit ○​ catalyzes formation of the peptide bonds that PROTEIN FACTORS link amino acid residues in a protein ​ Small Ribosomal Subunit ​ Initiation factors ○​ binds mRNA ​ elongation factors ○​ responsible for the accuracy of translation by ​ termination or release factors ensuring correct base-pairing between the ​ required for peptide synthesis codon in the mRNA and the anticodon of the ​ Functions: tRNA ○​ Catalytic ○​ stabilize synthetic machinery 1.​ rRNA ○​ have extensive regions of secondary ATP AND GTP AS ENERGY SOURCES structure arising from the base pairing of ​ cleavage of 4 high energy phosphate bonds for complementary sequences of nucleotides of the addition of 1 amino acid to the growing different portions of the molecule. polypeptide ○​ 2 from ATP in the aminoacyl-tRNA 2.​ Ribosomal Proteins synthetase reaction ○​ greater amount in eukaryotic than prokaryotic ​ 1 in PPi removal ribosomes ​ 1 in the subsequent PPi hydrolysis to ○​ role in the structure and function of the inorganic phosphate by ribosomes and its interactions with other pyrophosphatase components of the translation system. ○​ 2 from GTPs ​ 1 for binding the aminoacyl-tRNA to 3.​ A, P, and E Sites on the Ribosomes the A site ○​ binding sites for tRNA molecules ​ 1 for the translocation step ○​ together, they cover 2 neighboring codons ​ additional ATP and GTP molecules required for ​ Acceptor (A) Site initiation in eukaryotes -​ binds an incoming aminoacyl-tRNA ​ additional GTP molecule required for ​ Peptidyl (P) Site termination in both eukaryotes and - occupied by peptidyl-tRNA prokaryotes - tRNA carries the chain of amino acids that has already been V. AMINO ACID ACTIVATION: FORMATION synthesized OF AMINOACYL-tRNAs ​ Exit (E) Site - occupied by the empty tRNA as it is ADAPTOR MOLECULES about to exit the ribosome. ​ aminoacylated tRNAs (charged tRNAs) ○​ link between the message to protein ○​ tRNAs to which amino acid has been covalently attached AMINOACYL-TRNA SYNTHETASES ​ required for attachment of amino acids to their corresponding tRNAs ​ implement the genetic code ​ responsible for the activation and attachment of the amino acids to the 3’-terminal adenosine of their corresponding tRNAs Figure 15. Initiation of protein synthesis. P site, peptidyl site on the ribosome; A site, aminoacyl site on the ribosome; E site, free tRNA ejection site (the A, P, and E sites or portions of them are indicated MORPHOLOGY by the dashed lines); eIF, eukaryotic initiation factor. This is a simplified version of translational initiation because many more ​ vary in initiation factors and steps are required. ○​ molecular weight ○​ number of subunits Cellular Location of Ribosomes ○​ amino acid composition a.​Eukaryotic Cells i.​ free in the cytosol or in close association with the rough endoplasmic reticulum BATCH TANNAWAG 1E ​ 6 BIOCHEM LC 7: PROTEIN SYNTHESIS ​ Dr. JANDOC, B. 02/11/2025 SPECIFICITY ​ very specific in their attachment of the correct amino acid to the correct tRNA ​ codon recognition is entirely due to the tRNA → fidelity of protein synthesis depends largely on the high specificity of tRNA synthetases ○​ Selectivity ​ the very high specificity of tRNA synthetases ​ due to the high selectivity of the enzyme for the amino acid to be activated and tRNA to which the amino acid is to be attached ○​ Proofreading and Editing Capability ​ if incorrectly activated the wrong amino acid ​ adds the wrong amino acid to the tRNA → deactivate the amino acid by hydrolysis hydrolyze the amino acid from the tRNA CLASSES ​ two structurally unrelated classes of aminoacyl-tRNA synthetases: Class I & Class II Figure 26. Generation of the initiator N-formylmethionyl-tRNA (fMet-tRNA). ​ Differences ○​ mechanism by which they recognize their ANTIPARALLEL BINDING BETWEEN CODON tRNA substrate AND ANTICODON ○​ initial site of aminoacylation on the tRNA 1.​ Rules ○​ amino acid specificity ○​ complementarity ○​ antiparallelism AMINOACYLATION REACTION 2.​ Codon-Anticodon Pairing ​ attachment of an amino acid to its proper tRNA ○​ mRNA codon is read 5’ → 3’ by an ​ catalyzed by tRNA synthetases anticodon (of tRNA) pairing in the flipped (3’ 5’) orientation a.​Aminoacyl-Adenylate Complex Formation 3.​ Base Pairing ○​ 1st 2 bases of the codon and the last 2 bases of the anticodon ​ by normal base pairing: - guanine-cytosine (G-C) b.​Aminoacyl Group Transfer to the 2’ or - adenine-uridine (A-U) 3’-Hydroxyl Group of the 3’-Adenosine ○​ last base of the codon with the 1st base of the anticodon Aminoacyl-AMP + tRNA ↔ Aminoacyl-tRNA + ​ follows less rigid requirements AMP ​ allows some tRNAs to base pair with >1 codon c.​Sum of the Above Reactions 4.​ Writing the Sequences of Both Codons and Anticodons ○​ the nucleotide sequence must always be listed in 5’ 3’ order WOBBLE HYPOTHESIS d.​Pyrophosphate (PPi) Hydrolysis ​ mechanism by which tRNAs can recognize >1 ○​ forms 2 free phosphates codon for a specific amino acid ○​ makes the overall reaction irreversible ​ base at the 5’-end of the anticodon (1st base of ○​ 2 high-energy phosphate bonds are expended the anticodon) in the formation of a single aminoacyl-tRNA ​ not as spatially defined as the other 2 bases VI. CODON RECOGNITION by tRNA ​ movement (“wobble”) of the 1st base (CODON-ANTICODON RECOGNITION) - allows non-traditional base pairing ​ recognition of a particular codon in mRNA is with the 3’-base of the codon (last base accomplished by the anticodon sequence of of the codon) tRNA - allows a single tRNA to recognize >1 ​ some tRNAs recognize >1 codon for a given codon → wobbling → there need not be amino acid 61 tRNA species in order to read the 61 codons coding for amino acids BATCH TANNAWAG 1E ​ 7 BIOCHEM LC 7: PROTEIN SYNTHESIS ​ Dr. JANDOC, B. 02/11/2025 IF-3 70S ↔ 50S + 30S → 30S IF-3 + 50S IF-1 IF-3 - act as antiassociation factor IF-1 - increases the forward rate of reaction 5.​Formylmethionine-tRNAf (fmet-tRNAf) ​ tRNAf ​ initiator tRNA ​ brings modified methionine (fmet) to the 30S initiation complex ​ different sequence than the tRNA (tRNAm) that inserts methionine in internal positions of the peptide chain ​ Aminoacyl-tRNA Synthetase ​ links methionine to both tRNAf & tRNAm Figure 17. Wobble: Nontraditional base-pairing between the 5'-nucleotide (first nucleotide) of the anticodon with the 3'-nucleotide (last nucleotide) of the codon. H = hypoxanthine (the product of adenine deamination). RULES GOVERNING BASE PAIRING OF THE Figure 18. tRNA charging CODONS 3RD BASE POSITION ​ 1st base in the anticodon is cytosine or ​ Transformylase adenine → pairing in the 3rd base of the codon ​ adds formyl group from is normal N10-formyltetrahydrofolate to the ​ 3rd base would be guanine or uracil respectively amino group of methionine that is ​ 1st base in the anticodon is uracil → 3rd base of attached to the tRNAf → N-fmet-tRNAf the codon can be either of the purines (adenine or guanine) ​ 1st base in the anticodon is guanine → 3rd base of the codon can be either of the pyrimidines (uracil or cytosine) ​ 1st base in the anticodon is inosine (which is possible because tRNAs have many unusual bases) → 3rd base of the codon can be ○​ Adenine ○​ Cytosine ○​ Uracil VII. PROTEIN SYNTHESIS in PROKARYOTES ​ nucleotide sequence on the mRNA is translated into the language of amino acid sequence ​ mRNA is translated from 5’-end to 3’-end → synthesis of proteins from the amino-terminal end to the carboxyl-terminal end ​ eukaryotic protein synthesis resembles that of prokaryotes in most details INITIATION Figure 19. Generation of the initiator N- formyl-methionyl-tRNA (fMet-tRNA). THF= tetrahydrofolate, C= cytosine, A= adenine. 30S INITIATION COMPLEX FORMATION ​ 1st event in protein synthesis STEPS IN THE 30S INITIATION COMPLEX ​ Requirements FORMATION 1.​mRNA Strand – to be translated ​ 30S Subunit Binds to a Specific mRNA Site 2.​Initiation Factors – facilitate initiation complex ○​ in association with: IF-1, IF-2, IF-3 assembly ​ IF-1, IF-2, IF-3 i.​ IF-3 – required for binding mRNA to the 30S 3.​GTP – provides energy for the process subunit 4.​30S Ribosomal Subunit ii.​ 2 mechanisms by which the ribosomes ​ 70S Particle Dissociation recognizes the nucleotide sequence that initiates translation BATCH TANNAWAG 1E ​ 8 BIOCHEM LC 7: PROTEIN SYNTHESIS ​ Dr. JANDOC, B. 02/11/2025 ​ Shine-Dalgarno Sequence (in E. coli ​ To Complete 30S Initiation Complex ​ purine-rich sequence Formation ​ identical with or similar to the sequence ○​ fmet-tRNAf binds to the 30S particle 5’-UAAGGAGGU-3’ ○​ IF-2 ​ base pairs with a pyrimidine sequence in ​ in association with GTP the 16S rRNA ​ proper binding of the initiator tRNA to the ​ 16S ribosomal RNA component of the 30S particle 30S ribosomal subunit ○​ IF-3 ​ has a nucleotide sequence near its 3’-end ​ dissociates from the 30S initiation complex that is complementary to all or part of upon binding of the fmet-tRNAf the Shine-Dalgarno sequence ​ located 6-10 bases upstream of the IF-3 RELEASE initiator (AUG or GUG) codon on the ​ allows the 50S subunit to bind to the 30S mRNA molecule (near its 5’-end) initiation complex to form the 70S initiation ​ places the initiator codon in the proper complex position in the 30S subunit to bind to the initiator tRNA (fmet-tRNAf) → the mRNA a.​GTP Hydrolysis 5’-end and the 3’-end of the 16S ○​ → GDP + Pi upon formation of the 70S ribosomal RNA can form complementary initiation complex base pairs → facilitated binding and b.​Released from the 70S Initiation Complex positioning of the mRNA on the 30S ○​ IF-1, IF-2, GDP, and inorganic phosphate (Pi) ribosomal subunit c.​2 Sites of the 70S Ribosome that can be ​ when fmet-tRNAf is the initiator codon Occupied by Aminoacylated tRNAs ​ pairs with GUG (even though GUG is ○​ peptide (P) site & amino (A) site normally a valine codon) d.​P Site ○​ occupied by fmet-tRNAf Figure 20. Complementary binding between prokaryotic mRNA Shine- Dalgarno sequence and 16S rRNA ​ Initiation Codon ​ AUG Codon ​ at the beginning of the message ​ recognized by a special initiator tRNA ​ facilitated by IF-2 in E. coli ​ goes to the P site ​ all other charged tRNAs enter at the A site ​ In Bacteria and Mitochondria ​ initiator tRNA carries an N-formylated methionine ​ formyl group is added to methionine ​ after the amino acid is attached to the initiator tRN Figure 22. Initiation of translation in E.coli ​ by transformylase – uses N10-tetrahydrofolate as the carbon donor ​ N-terminal methionine is usually removed before the protein is completed Figure 23. Prokaryotic vs. Eukaryotic Translation ELONGATION ​ Aminoacylated tRNA ○​ complementary to the codon adjacent to the initiator codon (ex: AUG) ○​ inserted in the A site→ starting the process of elongation Figure 21. Initiation codon in bacteria and mitochondria. BATCH TANNAWAG 1E ​ 9 BIOCHEM LC 7: PROTEIN SYNTHESIS ​ Dr. JANDOC, B. 02/11/2025 a.​Elongation Factors Required i. Movement of the uncharged tRNA from ○​ EF-Tu, EF-Ts, EF-G the P site to the E site ○​ abundant proteins ii. Movement of the dipeptidyl-tRNA to the ○​ present in the cell at levels of 5-10% of all P site proteins iii. Unoccupied A site b. EF-Tu ○​ effects delivery of an aminoacyl-tRNA to the ○​ EF-G empty A site ​ catalyzes translocation c. GTP ​ forms a complex with GTP during ○​ bound to EF-Tu translocation ○​ hydrolyzed ​ GTP → GDP + Pi d. GDP ○​ from GTP hydrolysis ○​ remains associated with EF-Tu until displaced by EF-Ts e. EF-Tu and EF-Ts ○​ forms a complex that is split by the binding of another GTP → EF-Tu-GTP complex formation for the delivery of the next aminoacyl-tRNA f. EF-Tu-GTP ○​ delivers all aminoacyl-tRNA except fmet-tRNAf to the A site ​ Peptide Bond Formation ○​ Amino Acid Transfer ​ the activated amino acid attached to the tRNA in the P site, initially fmet-tRNAf, is transferred to the amino group of the aminoacyl-tRNA in the A site ​ addition of amino acids to the carboxyl end of the growing chain ○​ Peptidyl Transferase ​ catalyzes peptide bond formation ​ activity intrinsic to the 23S rRNA (ribozymes) found in the 50S ribosomal subunit ○​ Result of the Reaction ​ 2 amino acids being attached to the tRNA (dipeptidyl-tRNA) in the A site ​ leaves an uncharged tRNA in the P site Figure 25. Formation of a peptide bond. Involves transfer of the peptide on the transfer RNA (tRNA) in the A site (transpeptidation). Figure 24. Peptide bond formation. ​ Translocation ○​ Ribosomal Movement ​ moved 3 nucleotides in a 5’→ 3’ direction along the mRNA, results in the following: BATCH TANNAWAG 1E ​ 10 BIOCHEM LC 7: PROTEIN SYNTHESIS ​ Dr. JANDOC, B. 02/11/2025 ​ RF-1 – recognizes the termination codons: UAA & UAG ​ RF-2 - recognizes: UGA & UAA ​ RF-3 – binds GTP: stimulates the activities of RF-1 and RF-2 → promoting termination BINDING OF RELEASE FACTORS ​ induces peptidyl transferase to release the polypeptide from the tRNA in the P site by hydrolysis ​ tRNA is also released from the P site RIBOSOMAL SUBUNIT SEPARATION ​ GTP-hydrolysis-dependent ○​ 30S Subunit ​ may move along the mRNA until another Shine-Dalgarno sequence is encountered resumption of translation ​ may completely dissociate from the mRNA ENERGY REQUIREMENTS FOR PROTEIN SYNTHESIS ​ each peptide bond formed requires 5 (7) high energy phosphate bonds Figure 26. Steps in prokaryotic protein synthesis (translation), and their inhibition by antibiotics. [Note: EF-Ts is a guanine nucleotide exchange factor. It facilitates the removal of GDP, allowing its 1.​tRNA Aminoacylation replacement by GTP. The eukaryotic equivalent is EF-1βγ.] fMet = ○​ ATP → AMP + 2 phosphates for every amino formylated methionine; S = Svedberg unit; GTP = guanosine nucleoside triphosphate; Phe = phenylalanine. [Note: Ricin, a toxin acid attached to its cognate tRNA from castor beans, removes an A from the 28 S rRNA in the large 2.​fmet-tRNAf Binding to the P Site subunit of eukaryotic ribosomes, thereby inhibiting their function.] ○​ GTP → GDP + phosphate upon initiation of Lys = lysine; Arg = arginine. every polypeptide synthesized 3.​ Aminoacyl-tRNA Binding to the A Site ○​ GTP → GMP + 2 phosphate for every aminoacyl-tRNA bound to the A site 4.​Translocation ○​ GTP → GDP + phosphate for every translocation step 5.​Termination ○​ GTP → GDP + phosphate for every polypeptide synthesis terminated ROLE OF HIGH ENERGY REQUIREMENTS DURING TRANSLATION 1.​High Translation Fidelity ○​ protein synthesis in E. coli ○​ error frequency of 1 ​ are recognized by release factors ribosomes at a time translate the mRNA ​ RF-1 or RF-2 BATCH TANNAWAG 1E ​ 11 BIOCHEM LC 7: PROTEIN SYNTHESIS ​ Dr. JANDOC, B. 02/11/2025 ​ Initiation Factors ​ at least 10 initiation factors (eIF) Figure 32. Protein Factors Required for Initiation of Translation in Bacterial and Eukaryotic Cells ​ 40S Ribosomal Subunit Figure 29. Process of Protein Synthesis on an mRNA Strands ​ 60S Ribosomal Subunit – bound by eIF-6 → 60S subunit prevented from VIII. PROTEIN SYNTHESIS IN EUKARYOTES binding to the 40S subunit ​ eukaryotic protein synthesis resembles that of ​ 40S Ribosomal Subunit – bound by prokaryotes in most details eIF-4C role in the association of the 2 ​ the differences are a consequence of the more ribosomal subunits complex cellular organization of the eukaryotic cell INITIATION ​ same basic events occur ​ 40S Initiation Complex Formation ○​ Requirements ​ mRNA Strand – monocistronic ​ use only 1 initiator codon (AUG) ​ do not use Shine-Dalgarno sequence to direct initiation to the initiator codon ​ less rigidly defined sequences are known to be involved to direct Figure 33. 80S Ribosome Dissociation initiation to the initiator codon ​ have 7-methylguanylate cap on their ​ met-tRNAi 5’-ends role in the initiation of ​ initiator tRNA – carries unmodified translation methionine (not formylated) to the 40S initiation complex ​ 2 Structurally Different tRNAs – recognize AUG codons ○​ tRNAi – recognizes the initiator codon ○​ tRNAm – recognizes the internal non initiator methionine codons ​ Methionyl-tRNA Synthetase ○​ adds methionine residue to either ○​ tRNAi or tRNAm STEPS IN THE 40S INITIATION COMPLEX FORMATION ​ Preinitiation Complex Formation Ternary Complex + 40S Subunit → Preinitiation Complex ​ Ternary Complex ○​ formed between: eIF-2, GTP, & met-tRNAi ​ mRNA Binding to the Preinitiation Complex of the Ribosome Figure 31. 5’ and 3’ Modification of mRNA ○​ not entirely understood BATCH TANNAWAG 1E ​ 12 BIOCHEM LC 7: PROTEIN SYNTHESIS ​ Dr. JANDOC, B. 02/11/2025 ○​ exact events ○​ functions of the initiation factors ​ Initiation factors ​ all function during mRNA binding ​ includes: eIF-3, eIF-4A, eIF-4B, eIF-4C, eIF-4F ​ Eukaryotic Initiation Factor (eIF) 4A ​ includes a subunit that acts as a cap-binding protein (CBP) ​ CBP - may initiate mRNA binding by interacting with 5’-cap structure; promotes binding of eIF-4A and eIF-4B to mRNA ​ Importance of Cap – cap analogue 7-methylguanosine monophosphate is a potent initiation inhibitor ​ ATP – hydrolyzed and bound by eIF-4A → unwinding of the secondary structure in the 5’-untranslated region of the mRNA ​ 40S Initiation Complex Formed Figure 34. Prokaryotic and Eukaryotic Translation ​ Initiation Codon Recognition ○​ 5’-untranslated region of the mRNA ○​ variable length ○​ between 40-80 nucleotides (regions longer than 700 nucleotides are known to occur) ○​ no Shine-Dalgarno sequence is present ○​ less rigidly defined sequences are known to be involved in the process of initiator codon selection ​ i. Initiation -​ occurs at the 1st AUG codon from the 5’-end ii. Kozak Consensus Sequence -​ recognized by the ribosome as the translational start site (sequence is A or G - CCAUGG) -​ aids in defining the initial AUG codon for translation -​ loss of the sequence reduced efficiency of translational initiation iii. 40S Ribosomal Subunit -​ binds near the 5’-end of the mRNA Figure 35. Diagrammatic representation of the initiation phase of -​ scans the mRNA in the 3’-direction until it finds protein synthesis on an eukaryotic mRNA template containing a 5′ the 1st AUG in the proper sequence context cap (Cap) and 3′ poly(A) terminal [(A)n]. -​ ATP hydrolysis -​ essential for the scanning process ELONGATION ​ similar mechanism as in prokaryotes 80S INITIATION COMPLEX FORMATION ​ Necessary Components ○​ 80S Initiation Complex ​ requires the actions of: eIF-5 & eIF-4C ○​ aminoacyl-tRNA ○​ GTP 60S Subunit + 40S Subunit 80S Initiation Complex ○​ eukaryotic elongation factors – eEF-1ɑ, eEF-1β, eEF-2 ​ Before 60S Subunit Joins the 40S Subunit ​ Aminoacyl-tRNAs ○​ removed are: eIF-2GDP & eIF-3 ○​ bound to the P site as ternary complexes ○​ requires eIF-5 (eEF-1α GTP aminoacyl-tRNA) ​ After Release ○​ GTP hydrolysis → GDP ○​ eIF-2GDP → eIF-2GTP by the action of eIF-2B ​ Analogies ​ Upon Completion of 80S Initiation Complex ○​ eEF-1α to prokaryotic EF-Tu Formation ○​ eEF-1α to prokaryotic EF-Ts ○​ all other initiation factors are removed ○​ eEF-1β – catalyzes the GDP to GTP exchange in the eEF-1α ​ Peptide Bond Formation ○​ same mechanism as in prokaryotes ​ Translocation ○​ Requires: eEF-2 GTP complex & GTP hydrolysis ○​ analogous to prokaryotic translocation mediated by EF-G-GTP complex BATCH TANNAWAG 1E ​ 13 BIOCHEM LC 7: PROTEIN SYNTHESIS ​ Dr. JANDOC, B. 02/11/2025 cells of the blood vessels. The swelling and imbibition results in the rupture of those pericytes, thereby weakening the structure of blood vessels causing dilatation leading to retinal microaneurysms. There is a compensatory mechanism to restore blood supply to the distal ischemic areas through the expression of endothelial growth factors. These transcription factors will stimulate the DNA to cause proliferation and sprouting of new blood vessels. However, these new vessels from neovascularization are very weak that they easily rupture; so it can cause a vicious cycle. We don’t Figure 36. Elongation of a polypeptide chain want those new blood vessels, so we can edit the VEGF to render the VEGF mRNA useless therefore TERMINATION there will be no protein products. Or we can do it in ​ similar mechanism as in prokaryotes another way, we need erythropoietin to increase blood cell production. Increasing this activity will 1.​Single Release Factor (eRF) stabilize the mRNA so that it is constantly available ○​ with an associated GTP to produce factors. ○​ recognizes all 3 termination codons 2.​Peptidyl Transferase Binding of Regulatory Proteins to mRNA ○​ of the ribosome ○​ both eukaryotes and prokaryotes ○​ activity effects termination when the release ○​ Results – block translation & extend mRNA use factor binds to the ribosome by protecting it from degradation 3.​GTP Hydrolysis ○​ required for termination and dissociation of IX. PROTEIN LOCALIZATION IN EUKARYOTES the ribosomal subunits EUKARYOTIC PROTEIN SYNTHESIS Again, at the termination codon, there is no ​ all translation of eukaryotic nuclear genes corresponding tRNA therefore the release factor begins on ribosomes free in the cytoplasm recognizes that termination codon causes the ​ proteins translated on free cytoplasmic ribosomes release of polypeptide, the tRNA, and the ○​ Cytoplasmic Proteins dismantling of the 80s initiation complex. This is ○​ Mitochondrial Proteins energy dependent. ​ encoded by nuclear genes ​ proteins being translated may belong in other locations ​ certain proteins are translated on ribosomes associated with the rough endoplasmic reticulum 1.​ Secretory Proteins ○​ extracellular matrix protein collagen 2.​ Serum Proteins Figure 37. Prokaryotic Versus Eukaryotic Translation ○​ Immunoglobulins, peptide hormone, ​ & serum albumin GENE EXPRESSION REGULATION 3.​ Organelle Proteins ○​ as those of the mitochondria 1.​Transcriptional Level ○​ >90% have to be imported from the cytoplasm ○​ gene expression is most commonly regulated 4.​ Integral Membrane Proteins at this level ○​ have to be inserted into the correct 2.​Translational Level intracellular membrane with the correct ○​ rate of protein synthesis is also sometimes orientation to properly function regulated ○​ accomplished in eukaryotes is by covalent PROTEIN SYNTHESIS ON MEMBRANE-BOUND modification of eIF-2 (phosphorylated eIF-2 is RIBOSOMES inactive) ​ proteins synthesized in the endoplasmic reticulum are made in a precursor form that is processed We can block transcriptional and translational levels before they reach their final destination through editing the mRNA. Editing the mRNA can render it inactive or useless or if we don’t want the SIGNAL HYPOTHESIS effect of that DNA. ​ Leader or Signal Sequence ○​ sequence of the proteins to be made on the For example, a common complication of Diabetes is endoplasmic reticulum on their amino terminal accumulation of sorbitol in the retina causing end imbibition of water by pericytes at the structural BATCH TANNAWAG 1E ​ 14 BIOCHEM LC 7: PROTEIN SYNTHESIS ​ Dr. JANDOC, B. 02/11/2025 ○​ directs the protein synthesis in the endoplasmic reticulum ○​ usually 15-30 amino acids long ○​ contains a stretch of hydrophobic amino acids ​ Steps in the Leader Sequence Recognition and Direction of Protein Synthesis to the Endoplasmic Reticulum ○​ Signal Sequence Recognition by the Signal Recognition Particle (SRP) ​ SRP - elongated complex of Figure 38. Protein Synthesis on the RER ​ 6 non-identical proteins ​ small cytoplasmic RNA (scRNA) PROTEIN TARGETING ​ SRP-Ribosome Complex Binding to the ​ many proteins are destined to perform their Endoplasmic Reticulum and functions within specific cellular organelles Continuation of Protein Synthesis into ​ Amino Acid Sequences the Lumen of the Endoplasmic ○​ some proteins; direct proteins to their final Reticulum locations ​ The translation-arrested SRP-ribosome ○​ Nuclear Proteins – contain nuclear complex (docking proteins) interacts localization signal with a SRP receptor on the ○​ Mitochondrial Proteins – have mitochondrial endoplasmic reticulum entry sequence ​ SRP Receptor – made of 2 dissimilar, ​ Other Signals integral membrane proteins ○​ N-terminal Hydrophobic Signal Sequence ​ SRP-Ribosome Complex and SRP ​ ensure translation on the RER Receptor Interaction → signal ​ found on proteins destined to be sequence insertion into the membrane secreted (insulin), placed in the cell protein synthesis resumption membrane (Na+ -K+ ATPase), or ​ GTP Cleavage – just prior to the ultimately directed to the lysosome resumption of protein synthesis; by the (sphingomyelinase) SRP receptor → SRP dissociation from ​ Phosphorylation of Mannose Residues the ribosomes ○​ direct an enzyme to a lysosome ​ During signal sequence insertion into the ○​ Lysosomal Enzymes and Phosphorylation membrane – signal sequence of Mannose associates with another integral ​ lysosomal enzymes are glycosylated membrane protein (signal sequence and modified receptor) ​ Golgi apparatus → specific mannose ​ Completion of Synthesis into the residues located in N-linked Endoplasmic Reticulum oligosaccharide chains phosphorylated ○​ Signal Peptidase by N-acetylglucosamine-1 ​ on the luminal side of the endoplasmic phosphotransferase → reticulum (- while - the signal sequence is mannose-6-phosphate in the attached to the signal sequence receptor oligosaccharide chain → protein ​ remainder of the protein is being removed from the secretion pathway → synthesized) directed to lysosomes ​ cleaves the signal sequence from the protein ○​ I-Cell Disease ○​ Protein passes through the membrane into the ​ genetic defect affecting phosphorylation lumen during synthesis → lysosomal enzymes released into ○​ For Some Proteins the extracellular space → inclusion ​ integral membrane proteins may form a bodies accumulate in the cell → passage or channel through which they compromised function may pass during synthesis and enter the ​ Manifestations: lumen ​ coarse facial features, gingival ​ Signal Membrane Protein Synthesis hyperplasia, macroglossia ○​ similar except for stop-transfer signals ​ craniofacial abnormalities, joint ○​ Stop-Transfer Signals immobility, clubfoot, claw- hand, ​ sequence of hydrophobic amino acids scoliosis within the protein ​ psychomotor retardation, growth ​ halts the transfer of the protein across the retardation membrane ​ cardiorespiratory failure, death in first ​ function as membrane-binding sequence decade ​ bone fracture and deformities ​ mitral valve defect ​ secretion of active lysosomal enzymes into blood and extracellular fluid BATCH TANNAWAG 1E ​ 15 BIOCHEM LC 7: PROTEIN SYNTHESIS ​ Dr. JANDOC, B. 02/11/2025 FOLDING INTO TERTIARY AND QUATERNARY CONFORMATIONS 1.​Folding into Tertiary and Quaternary Conformations ○​ Proper Disulfide Bond Formation ​ important component in the formation and maintenance of the proper tertiary and quaternary conformations of many proteins ​ occurs in the lumen of the endoplasmic reticulum ​ catalyzed by protein disulfide isomerase ○​ Multimeric Protein Assembly ​ occurs in the lumen of the endoplasmic reticulum Figure 39.Children with I-cell disease ○​ Mutated Proteins ​ do not allow proper folding ​ often not transported from the endoplasmic reticulum ​ ex: 1-antitrypsin ​ major cause of emphysema in caucasians ​ protease inhibitor ​ produced in the endoplasmic reticulum of the liver and macrophages ​ as the neutrophils work in the lung neutrophils → elastase released → destroy lung cells ​ released elastase blocked by circulating α1-antitrypsin ​ single mutation → improper folding → Figure 40 Synthesis of Secretory, Membrane, and Lysosomal lack of antiprotease activity → Proteins development of emphysema caused by proteolytic destruction of lung cells → reduction in the expansion and contraction capability of the lungs 2.​Covalent Alterations ○​ Phosphorylation ​ occurs on hydroxyl groups of ​ Serine ​ threonine ​ tyrosine ​ catalyzed by protein kinases ​ reversed by protein phosphatases ​ may increase or decrease the functional activity of the protein Figure 41. Important Points About the Genetic Code, and Translation X. POSTTRANSLATIONAL MODIFICATION OF POLYPEPTIDE CHAINS Figure 42. Post Translational modification proteins POSTTRANSLATIONAL MODIFICATION OF POLYPEPTIDE CHAINS IN THE ENDOPLASMIC RETICULUM ​ covalent modifications either while still attached to the ribosome or after completion of synthesis BATCH TANNAWAG 1E ​ 16 BIOCHEM LC 7: PROTEIN SYNTHESIS ​ Dr. JANDOC, B. 02/11/2025 i. Solely N-glycosylated – transferrin ii. Solely O-glycosylated – heparin iii. Both N- and O-glycosylated – LDL receptor 4.​Hydroxylation ○​ Very important for collagen to attain its full enzyme strength ○​ in the endoplasmic reticulum ○​ proline and lysine residues of α-chains of collagen Figure 43. Phosphorylation of serine and tyrosine 3.​Glycosylation ○​ most proteins synthesized in the endoplasmic reticulum are glycosylated ○​ proteins destined to become ​ part of plasma membrane ​ secreted from the cell ○​ Have carbohydrate chains (oligosaccharides) Figure 45. Hydroxylation attached to: ​ hydroxyl groups (O-linked) of Serine and 5.​Other covalent modifications threonine ○​ for functional activity of a protein ​ amino group (N-linked) of asparagine ○​ Biotin – must be covalently bound to protein within the sequences: Asn-X-Ser & component of carboxylase enzymes to be Asn-X-Thr catalytically active ○​ core oligosaccharide ​ transferred as a complete unit from an ​ Carboxylation – carboxyl groups added to activated form glutamate residues by vitamin K-dependent ○​ activated core oligosaccharide carboxylation → 𝛄-carboxyglutamate ​ linked by a high-energy pyrophosphate residues (essential for the activity of several of linkage to a lipid donor molecule (dolichol) the blood-clotting proteins) within the endoplasmic reticulum membrane ​ Another modification will be the carboxylation ○​ glycosyl transferase of blood clotting factors so that we will not have ​ catalyzes the transfer bleeding tendencies or bleeding dialysis. ○​ much glycosylation (N-linked and O-linked) ​ occurs after proteins leave the endoplasmic reticulum and enter the Golgi apparatus ○​ N-linked glycosylation ​ begins in the endoplasmic reticulum as proteins are being synthesized ○​ Glycosylation may be O-linked or N-linked and this happens in the Endoplasmic reticulum and also in the Golgi apparatus. Figure 46. Carboxylation ​ Lipidation – farnesyl groups are very important because they help anchor proteins in the cell membrane. Figure 44. Glycosylation of serine and asparagine Figure 47. Farnesylated protein BATCH TANNAWAG 1E ​ 17 BIOCHEM LC 7: PROTEIN SYNTHESIS ​ Dr. JANDOC, B. 02/11/2025 ​ Acetylation – many proteins acetylated give you a 110 amino acids preproinsulin by the posttranslationally action of a signal peptidase - the signal ​ Biotinization - remember the biotin-dependent sequence made of 2 amino acids to be enzymes removed to produce a proinsulin - which is 86 amino acids and when it is cleaved by endo and exoproteases, it will remove the C peptide and an extra amino acid to produce an active insulin molecule. You’ll notice there, the intrachain disulfide bonding forming the insulin molecule into its functional form. ○​ synthesized in the β-cells of the islets of Langerhans in the pancreas ​ mRNA for Human Insulin – specifies 110 amino acids (preproinsulin) ​ Signal Sequence – formed by the 24 amino acids on the amino-terminal end; removed during synthesis in the endoplasmic reticulum ​ Proinsulin – formed by the remaining 86 amino acids and packed into secretory Figure 48. Biotinylated enzyme granules ​ Endoproteases and Exoprotease – within POSTTRANSLATIONAL MODIFICATION OF the secretory granules; hydrolyze bonds POLYPEPTIDE CHAINS BEYOND THE active insulin inactive C peptide 4 amino ENDOPLASMIC RETICULUM acids ​ Active Insulin – made of 21 amino acid A TRANSPORT TO THE GOLGI COMPLEX chain and 30 amino acid B chain; held ​ by vesicles together by disulfide bonds that were ​ Golgi Complex – principal director of the formed in the lumen of the endoplasmic intracellular movement of macromolecules reticulum because this is where the signal peptides are ​ Active Insulin and C Peptide – secreted in modified and produced. the plasma upon demand ​ signal for the movement are not well defined ​ responsible for the completion of the glycosylation of proteins that began in the endoplasmic reticulum TRIMMING OR PROTEOLYTIC CLEAVAGE ​ Molecules are activated and rendered functional after cleavage like for example: the zymogen, trypsinogen, when cleaved, it will be transformed into an active trypsin. ​ for the proteins to become active ​ Proteins Destined for Secretion from the Cell ○​ initially made as large, precursor molecules which are not functionally active ○​ portions removed by endoproteases → release of active molecule ○​ cellular site of the cleavage reaction depends on the protein to be modified Figure 50. Insulin biosynthesis ​ endoplasmic reticulum ​ Golgi apparatus ​ Glucagon ​ developing secretory vesicles (insulin) ○​ Is also synthesized as a precursor of the ​ some cleaved after secretion preproglucagon and by signal elastase and signal peptidase it will result to the formation of ​ Zymogens a proglucagon and another round of endo and ○​ inactive precursors of secreted enzymes exo proteolytic cleavage it will result into an ○​ activated through cleavage once they have active glucagon. reached their proper sites of action ○​ synthesized as a large preproglucagon in the ​ ex: Trypsinogen → trypsin in the small ɑ-cells of the islets of Langerhans in the intestines pancreas ○​ synthesized as zymogens to protect the cell ○​ Preproglucagon Signal Peptide from digestion by its own products ​ removed during synthesis in the endoplasmic reticulum proglucagon ​ Insulin ○​ Endoproteolytic Cleavages ○​ Insulin gene when expressed, the product will ​ within the secretory granules become an mRNA and when translated, it will ​ remove 29 amino acids (active glucagon) from within the proglucagon precursor BATCH TANNAWAG 1E ​ 18 BIOCHEM LC 7: PROTEIN SYNTHESIS ​ Dr. JANDOC, B. 02/11/2025 XI. DRUGS AND INHIBITORS OF PROTEIN SYNTHESIS IN EUKARYOTES THERAPEUTIC DRUGS STREPTOMYCIN ​ Prevents binding of a tRNA to the P site. ​ Aminoglycoside ​ used to treat heart infections, tuberculosis ​ prevents binding of fmet-tRNAf to the P site of the initiation complex → inhibits initiation of protein syn