SBP3411 Eukaryotic Protein Expression PDF 2023

SBP3411 Protein Synthesis in Eukaryotic Cells Dr. Hanis H. Harith Dept. of Biomedical Science, UPM [email protected]  Learning Outcomes At the end of this lecture the student is able to: Describe the genetic code Describe the structure and the role of tRNA, amino-acyl s...

SBP3411 Protein Synthesis in Eukaryotic Cells Dr. Hanis H. Harith Dept. of Biomedical Science, UPM [email protected]  Learning Outcomes At the end of this lecture the student is able to: Describe the genetic code Describe the structure and the role of tRNA, amino-acyl synthetase and ribosome in eukaryotic protein translation Describe key events in protein synthesis in eukaryotic cells Describe mechanisms that can regulate the final functional protein concentration Hanis Harith SBP3411 (2023) Dept of Biomedical Science UPM Lecture Outline The genetic code Key molecules in protein synthesis Protein synthesis Regulation of final protein concentration Hanis Harith SBP3411 (2023) Dept of Biomedical Science UPM Protein expression depends on regulation of mRNA expression and breakdown of protein Alberts et al. (2019). Essential Cell Biology (5th Edition) mRNA will be translated if not degraded Hanis Harith SBP3411 (2023) Dept of Biomedical Science UPM  The genetic code Codon sets of 3 nucleotides in mRNA matches with specific amino acid (aa) 1 aa may match with >1 codon 20 aa in total Applicable to most organisms slight differences in mitochondria Essential Cell Biology, Fifth Edition Hanis Harith SBP3411 (2023) Dept of Biomedical Science UPM Copyright © 2019 W. W. Norton & Company  Possible reading frames in mRNA translation Depends on where the reading frame starts (5’– 3’) 3 possible reading frames Only one encodes the correct protein An error will cause misreading of subsequent codons May result in synthesis of a nonfunctional protein Determined by the translation start signal or AUG codon Essential Cell Biology, Fifth Edition Hanis Harith SBP3411 (2023) Dept of Biomedical Science UPM Copyright © 2019 W. W. Norton & Company The role of tRNA in translation Adaptor molecules that match aa to codons in mRNA Aa is covalently attached at the 3’ end about 80 nt long Form a cloverleaf structure due to internal base-pairing, further fold into an L-shaped structure due to hydrogen bonding between different regions Anticodon base-pairs with complementary codon (Many tRNAs can tolerate a mismatch at 3rd position) Essential Cell Biology, Fifth Edition Hanis Harith SBP3411 (2023) Dept of Biomedical Science UPM Copyright © 2019 W. W. Norton & Company Aminoacyl-tRNA synthetases Catalyzes the formation of covalent bonds between tRNA and the correct aa Charged Product = charged tRNA tRNA Each recognizes a specific aa 20 synthetases in total Has a binding site for the specific aa, matching tRNA & ATP required for the reaction Makes contact with nucleotides in the anticodon loop and in the amino-acid- accepting arm that are specific to the correct tRNA Essential Cell Biology, Fifth Edition Hanis Harith SBP3411 (2024) Dept of Biomedical Science UPM Copyright © 2019 W. W. Norton & Company Charging of tRNA molecules Catalyzed by aminoacyl-tRNA synthetases Formation of a covalent bond between the specific aa to a matching tRNA Reaction coupling with ATP hydrolysis (which route?) The high-energy bond is used to link aa covalently to a growing polypeptide chain Determined by base-pairing between anticodon of charged tRNA and codons An error can result in incorporation of incorrect aa Essential Cell Biology, Fifth Edition Copyright © 2019 W. W. Norton & Company Hanis Harith SBP3411 (2024) Dept of Biomedical Science UPM Translation occurs on ribosomes Ribosomes are located in the cytoplasm; free or attached to ER A large complex consists of rRNA molecules and ribosomal proteins Small subunit: matches tRNA to codons Large subunit: 23S rRNA forms the catalytic site aka peptidyl transferase (ribozyme) that catalyze formation of peptide bonds by precisely orienting the growing polypeptide and aa carried by incoming tRNA rRNAs are positioned at the center and folded into a compact 3D structure Small subunit Ribosomal proteins mainly facilitate folding contains the and stabilize the RNA core binding site Movie 7.9 ECB for mRNA Both subunits assemble at the 5’ end of mRNA to form the 3 tRNA binding sites (A; Modified from Essential Cell Biology, Fifth Edition aminoacyl-tRNA, P;peptidyl-tRNA, E; exit) Hanis Harith SBP3411 (2023) Dept of Biomedical Science UPM  Additional proteins bound at the 5’ cap and Protein synthesis: Initiation poly-A tail facilitate the recognition of intact mRNA by translational apparatus (not shown) Requires assembly of several factors on small ribosomal subunit an initiator tRNA translation initiation factors (TIFs) Initiator tRNA charged with methionine; distinct from tRNA that normally carries methionine can bind tightly to P site in absence of large ribosomal subunit all newly made proteins start with methionine at the N-terminus (where protein synthesis starts); usually removed later by a specific protease Loaded small ribosomal subunit binds to 5’ end of mRNA (indicated by 5’ cap) scans mRNA (5’ to 3’) and stops once it reaches the first AUG codon Hanis Harith SBP3411 (2023) Dept of Biomedical Science UPM  Protein synthesis: Initiation (continued) Recognition of AUG leads to dissociation of some TIFs The assembly of ribosome is complete when the large ribosomal subunit binds to the complex Protein synthesis starts with the addition of the next charged tRNA to the A site Hanis Harith SBP3411 (2023) Dept of Biomedical Science UPM  Protein synthesis: Elongation A 4-step cycle which is repeated to complete protein synthesis Step 1: a new charged tRNA binds to the vacant A site (blue) determined by base-pairing with the complementary codon Step 2: Formation of peptide bond between new aa and growing polypeptide chain the carboxyl end of the growing peptide chain is uncoupled from the tRNA at the P site (orange) the last aa in the chain is linked to the free amino group of the new aa by a covalent bond aa is always added to the carboxyl end of the polypeptide chain (structural polarity) Essential Cell Biology, Fifth Edition Copyright © 2019 W. W. Norton & Company Hanis Harith SBP3411 (2023) Dept of Biomedical Science UPM  Protein synthesis: Elongation (continued) Step 3: Translocation of large subunit the large subunit shifts forward spent tRNA is moved to the E site & the new tRNA (holding the growing polypeptide chain in place) is moved to the P site Step 4: Translocation of small subunit the small subunit moves exactly 3 nt along the mRNA molecule results in subsequent ejection of the spent tRNA The final step resets the ribosome with an empty A site for the binding of the incoming charged tRNA molecule Movie 7.8 ECB Hanis Harith SBP3411 (2023) Dept of Biomedical Science UPM Protein synthesis: Termination Stop codons Termination signal for translation UAA, UAG, UGA codons are not recognized by tRNA or specify any aa Release factors bind to a stop codon that reaches the A site Peptidyl transferase activity is altered to catalyze addition of H2O instead This frees the carboxyl end of the polypeptide chain from attachment to a tRNA molecule and it is immediately released Finally, ribosome releases mRNA and dissociates They can assemble at another mRNA to initiate translation Essential Cell Biology, Fifth Edition Hanis Harith SBP3411 (2023) Dept of Biomedical Science UPM Copyright © 2019 W. W. Norton & Company Proteins are synthesized on polyribosomes Also known as polysomes Large cytosolic assemblies made up of multiple ribosomes spaced as close as 80 nucleotides apart along a single mRNA molecule Allows efficient protein synthesis of a single mRNA at a given time Movie 7.10 ECB Essential Cell Biology, Fifth Edition Hanis Harith SBP3411 (2023) Dept of Biomedical Science UPM Copyright © 2019 W. W. Norton & Company Post-translational processing of synthesized proteins Polypeptides synthesized may require further processing to become fully functional Fold correctly to gain the accurate 3D shape (spontaneously or facilitated by chaperone proteins) Binding to other factors e.g. small molecules, other protein subunits (non-covalent interactions) Undergo post-translational modifications (PTMs) that involve covalent modification (>100 types e.g. phosphorylation, glycosylation) PTMs can regulate the activity of proteins, determine their location and association with other factors Cells can regulate the final concentration of specific protein by controlling any of these steps Essential Cell Biology, Fifth Edition Copyright © 2019 W. W. Norton & Company Hanis Harith SBP3411 (2023) Dept of Biomedical Science UPM Protein concentration can be regulated by proteolysis Short-lived, damaged and misfolded proteins are tagged for destruction by covalent attachment of a short ubiquitin chain (ubiquitylation) Proteolysis: Breakdown of proteins into their constituent aa Proteases: Enzymes responsible for proteolysis by hydrolyzing peptide bonds Proteasomes: Large protein machines located in cytosol and nucleus The central cylinder is lined with proteases (active sites facing into an inner chamber) The stoppers at each end are large protein complex that bind proteins destined by degradation (ubiquitylated proteins), unfold and thread them into the inner chamber (requires ATP hydrolysis) Essential Cell Biology, Fifth Edition Hanis Harith SBP3411 (2023) Dept of Biomedical Science UPM Copyright © 2019 W. W. Norton & Company Protein expression depends on regulation of mRNA expression and breakdown of protein Alberts et al. (2019). Essential Cell Biology (5th Edition) Identify examples of control mechanisms at each of the steps involved in protein expression Hanis Harith SBP3411 (2023) Dept of Biomedical Science UPM