Protein Synthesis - Ch 32 - Fall 2023 - PDF

Protein Synthesis – Ch 32 CMB 704/DENT 604 – Fundamental Biochemistry Dr. Maryam Syed [email protected] Fall 2023 1 Learning Objectives 1. Define the terms initiation (or start) codon and termination (or stop) codon and identify their sequence. 2. List the components required for translation and s...

Protein Synthesis – Ch 32 CMB 704/DENT 604 – Fundamental Biochemistry Dr. Maryam Syed [email protected] Fall 2023 1 Learning Objectives 1. Define the terms initiation (or start) codon and termination (or stop) codon and identify their sequence. 2. List the components required for translation and summarize the role of each. 3. Explain the role of the messenger RNA (mRNA) cap and poly-A tail (euk) and role of Shine-Dalgarno (prok) in translation initiation. 4. Summarize the steps in the initiation, elongation, and termination of protein translation and include the protein factors involved. 5. Describe the mechanism by which microRNAs (miRNAs) function in regulation of translation. 6. Explain how frameshift, missense, and nonsense gene mutations alter the reading frame of the mRNA made from the gene and how these mutations will alter the protein synthesized from the mRNA. 7. Recall how secreted proteins are targeted to their final location. 8. Describe the major steps in the degradation of a cytosolic protein. 2 Learning Objectives Define the terms initiation (or start) codon and termination (or stop) codon and identify their sequence. 1. • Initiation/start codon: • • • 3bp sequence on mRNA that signals the start of protein synthesis; codes for Met; first aa in the peptide chain AUG Termination/stop codons: • • 3bp sequence on mRNA that signals the end of proteins synthesis; does not code for an aa; will be in the A site of the ribosome and will be bound/recognized by RF 1/2 (prok) and eRF(euk). UAA, UAG, UGA 3 Learning Objectives 2. List the components required for translation and summarize the role of each. • Amino acids: substrate added to growing peptide chain to make the nascent protein. • tRNA: adaptor molecule that carries appropriate aa to the site of synthesis on the ribosomal complex • Aminoacyl-tRNA synthetases: enzyme; catalyzes the attachment of aa to tRNA • Messenger RNA: carries the code in triplicate (3bp = 1 codon) • Functionally competent ribosomes: rRNA + large ribsome subunit +small ribosome subunit. • Protein factors: catalytic or structural roles to help in the process of translation 4 Lecture Name Learning Objective Difficulty Level Easy Medium Hard Bloom’s Level Recall Apply Synthesize Slide # Textbook pg # Web resources/links Key terms/Definitions Lecture Notes Summary Learning Strategy Elaboration Dual Coding Concrete Examples Anticipated Test Questions 5 Major Topics • Genetic code • Components required for translation • Codon recognition by tRNA • Steps in translation 6 Protein Synthesis Overview • DNA  RNA  Protein • Proteome = complete set of proteins expressed in a cell • Translation is the synthesis of proteins • Translation requires a genetic code • • Alterations in nt sequence = insertion of incorrect aa • • information in nt sequence produces aa sequence disease or death Nascent proteins undergo modifications and are folded to reach functional form 7 Genetic Code Codons Codon characteristics Consequences of altered nt sequences 8 Codons • 20 amino acids (aa) in proteins • Codons are in RNA language = A, G, C, U • Codon consists or 3 bases (nt) = triplet code • 43 = 64 combinations to code for 20 aa • Table used to translate a codon to determine which aa is coded by the triplet code on mRNA sequence 9 Initiation and Termination Codons One codon codes for initiation/start codon: 1. AUG Three codons code for termination/stop codon: 1. UAA 2. UAG 3. UGA 10 Genetic Code Characteristics Specificity • Genetic code is specific • A specific codon will always code for the same aa Universality • Specificity of genetic code is conserved through evolution 11 Genetic Code Characteristics Degeneracy • Genetic code is redundant • Each codon corresponds to a single aa • Single aa can be coded for by more than one codon 12 Genetic Code Characteristics Nonoverlapping and commaless • Genetic code is read from a fixed starting point • Read as 3 bases at a time AGCUGGAUACAU AGC / UGG / AUA / CAU 13 Consequences of Altering the Nucleotide Sequence • Point mutation (change in only one nt) in coding region of mRNA can cause: 1. Silent Mutation 2. Missense Mutation 3. Nonsense Mutation 14 Silent Mutation • Codon with point mutation codes for the same aa • Due to genetic code degeneracy 15 Missense Mutation • Codon with point mutation codes for a different aa 16 Nonsense Mutation • Codon with point mutation codes for a termination/stop codon • Introduces premature termination of translation • Remaining genetic code downstream of nonsense mutation is not translated 17 Trinucleotide Repeat Mutations Trinucleotide repeat expansion • 3 bases of codon repeated in tandem  many consecutive copies of triplet • Leads to insertion of consecutive extra copies of one aa in the protein • Over 20 triplet expansion diseases 18 Splice Site Mutations Normal Length Protein Splice Site Mutations • Mutation at splice site  aberrant protein produced Aberrant? Truncated? Truncated/Aberrant Protein due to splice site mutation that resulted in loss of an exon or coding region19 Frameshift Mutations Frameshift Mutations 1 or 2 nt added or deleted on coding mRNA 1. • Alteration of reading frame • • Product will be completely different aa sequence Truncated product due to premature introduction of termination/stop codon 2. 3 nt insertion  insertion of new aa in peptide 3. 3 nt deletion  deletion of aa in peptide What determines the reading frame? 20 Components Required for Translation 1. Amino acids 2. tRNA 3. Aminoacyl-tRNA synthetases 4. Messenger RNA 5. Functionally competent ribosomes 6. Protein factors 21 1. Amino Acids • All 20 aa must be present for protein synthesis to begin • Translation stops if an aa is missing 22 2. Transfer RNA • ~50 species in humans • ~30 species in bacteria • At least one specific tRNA required for each aa • Some aa have more than one specific tRNA 23 2. Transfer RNA Amino acid attachment site • Specific attachment site of cognate aa at 3’-end • aa is bound to A nt in –CCA sequence at 3’end of tRNA • tRNA with covalently attached aa = charged • tRNA without aa = uncharged 24 2. Transfer RNA Anticodon • tRNA contains 3 nt sequence = anticodon • tRNA anticodon pairs with complementary codon on mRNA 25 3. Aminoacyl-tRNA synthetases • Family of 20 enzymes required for attaching aa to tRNA • Each member recognizes specific aa and all corresponding tRNAs • High fidelity translation  Extreme specificity of synthetase in recognizing aa and cognate tRNA • Editing activity  removes incorrect aa from enzyme or tRNA Mechanism • Aminoacyl-tRNA synthetase = E 1. aa + E + ATP  E-AMP-aa 2. E-AMP-aa + tRNA  aa-tRNA 26 4. Messenger RNA • Required to translate desired polypeptide • Eukaryotic mRNA is circularized for use in translation 27 5. Functionally competent ribosomes • Large protein complex + rRNA • Site of protein synthesis • 2 subunits (large and small) • Similar structure and function in euk and prok • Small ribosomal subunit binds mRNA • Ensures correct binding between codon (mRNA) and anticodon (tRNA) • Large ribosomal subunit catalyzes peptide bond formation that links aa chain • 50S + 30S = 70S ? 28 5. Functionally competent ribosomes E, P, A sites • 3 binding sites for tRNA molecules • Cover 3 consecutive codons (9 bp) Translation • A (acceptor) site binds incoming aminoacyl-tRNA that corresponds to codon in that site • P (peptidyl) site occupied by peptidyl-tRNA (carries aa chain already synthesized) • E (empty) site occupied by empty (used) tRNA that will exit ribosome: • tRNA was first in A, then moved to P, then moved to E as ribosome moved 3 bases along mRNA 29 5. Functionally competent ribosomes Cellular location – Eukaryotes • Free Ribosomes in cytosol synthesize proteins to be used within the cell (cytosol, nucleus, mito) • RER-associated (bound) ribosomes synthesize proteins to be used outside of the cell (membrane, lysosomes) 30 6. Protein factors • Initiation, elongation, termination factors required for polypeptide synthesis • Various roles: 1. Catalytic 2. Stabilization of translation machinery 31 Codon Recognition by tRNA Antiparallel binding between codon and anticodon Wobble hypothesis 32 Antiparallel binding between codon and anticodon • mRNA codon is in 5’  3’ orientation • tRNA anticodon pairs in opposite 3’  5’ orientation 33 Wobble hypothesis • Allows tRNA to recognize more than one codon for specific aa • Codon-anticodon pairing follows traditional Watson-Crick rules for 1st and 2nd base of codon but 3rd (3’-end) can be less stringent • Base at 5’-end of anticodon (1st base) allows nontraditional base-pairing with 3’-base of codon (3rd base) 34 Steps in Translation 1. Initiation 2. Elongation 3. Termination 4. Regulation 5. Protein folding 6. Protein targeting 7. Protein degradation 35 Translation overview 1) How many start and stop codons are in this Euk mRNA? • mRNA is translated 5’  3’ • Protein synthesized from N-terminal to C-terminal • Each coding region of polycistronic (prok) mRNA has its own initiation and termination codon • • Coding region of monocistronic (euk) mRNA has just one initiation and termination codon • • Produces a separate species of polypeptide for each cistron Produces one polypeptide Translation in euk and prok similar with some 2) How many start and stop codons are in this Prok mRNA? exceptions 36 Initiation • Assembly of translation components 1. 2. 3. 4. 5. 6. 2 ribosomal subunits  makes up the functional ribosome complex; site of translation mRNA  contains code to make protein Aminoacyl-tRNA  carries aa (adaptor molecule) GTP  (e)IF-2-GTP help tRNAi find AUG start codon Initiation factors  facilitate assembly of initiation complex ATP (eukaryotes only)  needed for 40S to scan mRNA for AUG start codon • 3 IFs in prokaryotes – IF-1, IF-2, IF-3 • Many eIFs in eukaryotes 37 Initiation • Mechanisms by which ribosome recognizes nt sequence (AUG) to initiate translation differ 1. Shine-Dalgarno sequence – prokaryotes 2. 5’-cap – eukaryotes 38 Initiation Shine-Dalgarno sequence (SD; E. Coli) 1. • Purine-rich sequence on mRNA • Located slightly upstream of initiating AUG codon • 16S rRNA of small ribosomal subunit (30S) has nt sequence at 3’-end that is complementary to all or part of SD sequence near 5’-end of mRNA • Positions 30S close to AUG (start) codon on mRNA 39 Initiation 5’-Cap (eukaryotes) 2. • Small (40s) ribosomal subunit binds close to 5’cap of mRNA • • Scans mRNA 5’  3’ to find start codon (_____) • • Facilitated by eIF-4 proteins Requires ATP Checking mechanism in Euk: • • Cap-binding eIF-4 proteins interact with polyAtail binding proteins on mRNA to mediate circularization of mRNA  prevents incompletely processed mRNA from being translated Incomplete processing? When does this happen? 40 Initiation codon • Initiation AUG recognized by special initiator tRNA = tRNAi • Facilitated by IF-2-GTP (prok) and eIF-2-GTP (euk) • Only (e)IF-2 can recognize charged tRNAi • tRNAi is the only tRNA to go directly to P-site (instead of what site?_________) • Base modifications distinguish tRNAi from internal AUG-tRNA 41 Initiation codon • Bacterial tRNAi carries N-formylated methionine (fMEt) Eukaryotic tRNAi carries Met (not fMet) • Large subunit joins complex after charged tRNAi binds at P-site • • Premature binding of large subunit prevented by IF-3 (prok) and eIF-3 (euk) 42 Overview of steps for Initiation 1) P: 16S rRNA in 30S binds SD sequence of mRNA E: 40S + eIF-2 bind near 5’-cap of mRNA 2) P: tRNAi charged with fMet E: tRNAi charged with Met 3) P: fMet-tRNAi + IF-2-GTP locate AUG start codon E: Met-tRNAi + eIF-2-GTP locate AUG start codon 4) P: large ribosome subunit (50S) joins complex + fMet-tRNAi in P-site E: large ribosome subunit (60S) joins complex + Met-tRNAi in P-site 43 Elongation • Addition of aa to carboxyl end of growing peptide chain • P: EF-Tu-GTP and EF-Ts facilitate delivery of charged tRNA for next mRNA codon located in A-site • E: EF-1α-GTP and EF-1βγ facilitate delivery of charged tRNA for next mRNA codon located in A-site • rRNA in large subunit will catalyze via peptidyltransferase peptide bond formation between α-carboxyl group of aa in P site and the α-amino group of the aa in A site • This rRNA is a ribozyme  RNA with catalytic activity 44 Elongation • After peptide bond formation, peptide on tRNA at P-site transferred to aa on tRNA at A-site  transpeptidation • Ribosome advances 3nt along mRNA (toward 3’-end)  translocation • • P: requires EF-G-GTP E: requires eEF-2-GTP • Uncharged (empty/used) tRNA moved from P to E site for release • Peptidyl-tRNA moved from A to P site • Process repeated until termination codon encountered 45 Overview of Elongation 1. Charged tRNA delivered to A-site on ribosome complex 2. Ribozyme in large subunit catalyzes peptide bond between aa in P-site and aa in A-site 3. Transpeptidation: peptide on tRNA at P-site transferred to aa on tRNA at A-site 4. Translocation: P-site uncharged tRNA moved to E-site for release A-site peptidyl-tRNA moved to P-site 5. A-site ready to accept next charged tRNA 6. Repeat steps 1-5 until termination/stop codon. Which site would this stop codon have to be in (E, P, A)? 46 Termination • Occurs when one of the three termination codons moves into the A site • P: Stop codon recognized by RF-1 (UAA, UAG) and RF-2 (UGA) • E: All stop codons recognized by eRF • eRF / RF-1 or 2 binds to A-site (instead of tRNA)  hydrolysis of peptide bond linking peptide to tRNA in P-site  nascent peptide released • eRF-3 / RF-3-GTP releases eRF / RF-1 or 2 from A-site • Newly synthesized (nascent) peptide may undergo further modification • Ribosomal subunits, mRNA, tRNA, and protein factors recycled and used to synthesize another polypeptide 47 48 49 Protein factors in 3 stages of translation 50 Translation regulation • Gene expression most commonly regulated at transcription • • Why? Translation requires a lot of energy (ATP/GTP) Gene expression can be regulated at translation • E: Phosphorylation of eIF-2  inactivated • • What does eIF-2 do? Brings tRNAi to P-site P + E: proteins can bind mRNA and block translation  RISC • RNA-induced silencing complex  microRNA (small noncoding RNA) binds to mRNA and blocks ribosome assembly or clearance 51 Protein folding • Nascent peptide sequence must be folded  into functional, native state • Folding can be: 1. 2. Spontaneous Mediated by chaperone proteins What determines how a protein will fold? Primary linear amino acid sequence 52 Protein folding • Linear polypeptide folding based on aa side chain interactions • Secondary structures driven by hydrophobic effect • • α-helix, β-sheet, and β-turn Tertiary structures (domains) formed by stabilizing interactions • Disulfide bonds, hydrophobic interactions, hydrogen bonds, ionic interactions 53 Protein targeting How do proteins made in the cytoplasm go to where they are needed to function? • Proteins have aa sequences that direct them to their final locations • Nuclear proteins contain nuclear localization signal • Mitochondrial matrix proteins contain an N-terminal, mitochondrial entry sequence 54 Protein degradation • Defective, misfolded proteins, or rapid-turnover  marked for destruction by ubiquitination 1. Protein tagged with chain of ubiquitins 2. Ubiquitinated protein recognized by cytosolic proteasome 3. Proteosome unfolds, de-ubiquinates, and transports protein through its proteolytic core 4. Results in peptide fragments that are degraded to aa 55 Summary • Translation is the process of translating the sequence of nucleotides in mRNA to an amino acid sequence of a protein. • Translation proceeds from the amino to the carboxyl terminus, reading the mRNA in the 5′-to-3′ direction. • Protein synthesis occurs on ribosomes. • The mRNA is read in codons, sets of three nucleotides that specify individual amino acids. • AUG, which specifies methionine, is the start codon for all protein synthesis. • Specific stop codons (UAG, UGA, and UAA) signal when the translation of the mRNA is to end. • Amino acids are linked covalently to tRNA by the enzyme aminoacyl-tRNA synthetase, creating charged tRNA. • Charged tRNAs base-pair with the codon via the anticodon region of the tRNA. • Protein synthesis is divided into three stages: initiation, elongation, and termination. • Multiprotein factors are required for each stage of protein synthesis. • Proteins fold as they are synthesized. • Specific amino acid side chains may be modified after translation by a process known as posttranslational modification. • Mechanisms within eukaryotic cells specifically target newly synthesized proteins to different compartments in the cell. • Proteins are marked for degradation via a ubiquitination event. 56