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Protein Synthesis Chapter 17 Protein synthesis DNA Responsible for hereditary information DNA divided into genes Gene: Sequence of nucleotides Determines amino acid sequence in proteins Genes provide information to make proteins Protein synthesis DNA RNA protein Protein Synthesis Gene Expression: Process by which DNA directs the synthesis of proteins 2 stages Transcription Translation Protein synthesis Transcription: DNA sequence is copied into an RNA Translation: Information from the RNA is turned into an amino acid sequence Protein synthesis DNA RNA Transcription Protein Translation Protein Synthesis Central Dogma Mechanism of reading & expressing genes Information passes from the genes (DNA) to an RNA copy Directs sequence of amino acids to make proteins Protein synthesis Beadle & Tatum Bread mold 3 enzymes to make arginine Mutated mold’s DNA Mutated code for enzymes Unable to code for arginine Results Table Precursor Enzyme A Wild type Minimal medium (MM) (control ) Ornithine Enzyme B Citrulline Arginine Growth: Wild-type cells growing and dividing Control: Minimal medium No growth: Mutant cells cannot grow and divide Condition Enzyme C Classes of Neurospora crassa Class I Class II mutants mutants Class III mutants MM + ornithin e MM + citrullin e MM + arginine (control ) Summar y of results Gene (codes for enzyme) Can grow with or without any supplements Can grow on ornithine, citrulline, or arginine Class I mutants (mutation in gene A) Precurso r Enzyme Can grow Require only arginine on citrulline to grow or arginine Class II Class III mutants mutants (mutation in (mutation in gene C) gene B) Precurso Precurso r r Enzyme Enzyme Gene A Wild type Precurso r Enzyme A A A A Gene B Ornithin e Enzyme Ornithin e Enzyme Ornithin e Enzyme Ornithin e Enzyme B B B B Gene C Citrullin e Enzyme C Arginin e Citrullin e Enzyme C Arginin e Citrullin e Enzyme C Arginin e Citrullin e Enzyme C Arginin e Protein synthesis Beadle & Tatum One gene one enzyme One gene one protein One gene one polypeptide An albino racoon Cracking the code Codons (Triplet code)-mRNA Each codon corresponds to an aa 20 amino acids 64 triplet codes (codons) 61 code for aa-3 are stop codons Wobble: Flexible base pairing in the 3rd position 3’ end Cracking the code Reading frame Reading symbols in correct groupings 1 or 2 deletions or additions Gene was transcribed incorrectly 3 deletions Reading frame would shift Gene was transcribed correctly WHYDIDTHEREDCATEATTHEFATRAT WHYIDTHEREDCATEATTHEFATRAT WHYDTHEREDCATEATTHEFATRAT WHYTHEREDCATEATTHEFATRAT Cracking the code Universal code AGA codes for amino acid Arginine Humans & bacteria Genes from humans can be transcribed by mRNA from bacteria Produce human proteins Insulin RNA RNA (ribonucleic acid) Single strand Sugar –ribose (-OH on 2’ carbon) Uracil instead of thymine RNA mRNA: Messenger RNA Transcribes information from DNA Codons (3 nucleotides) CGU mRNA Codes for amino acids rRNA: Ribosomal RNA Polypeptides are assembled RNA tRNA: Transfer RNA Transports aa to build proteins Positions aa on rRNA Anticodons (3 complementary nucleotides) GCA Nuclear envelope TRANSCRIPTION DNA PreRNA PROCESSING mRNA NUCLEUS TRANSCRIPTION CYTOPLASM DNA mRNA Ribosome CYTOPLASM TRANSLATION TRANSLATION Ribosom e Polypeptide Polypeptide (a) Bacterial cell mRNA (b) Eukaryotic cell Transcription Getting the code from DNA Triplet code Template strand Strand of DNA Provides template or pattern Transcribed or read Transcribed RNA is complementary to this DNA strand Transcription Coding strand DNA strand not coded Same sequence of nucleotides as the RNA transcript Only T instead of U. Figure DNA 17.4 templat e strand 3′ A C C A A A C C G A G T T G G T T T G G C T C A 5′ 5′ 3′ TRANSCRIPTIO N mRNA 5′ TRANSLATIO N Protei n U G G U U U G G C U C A Codo n Trp Amino Phe Gly Ser 3′ Transcription RNA polymerase Enzyme Adds nucleotides to the 3’end 5’to3’ direction Does not need a primer to start One polymerase in prokaryotes Three in eukaryotes Polymerase II makes mRNA Transcription Promoters: Sequence on DNA where transcription starts TATAAT TATA box Sequences are not transcribed Transcription Stages Initiation Elongation Termination Initiation RNA polymerase binds promoter Unwinds DNA Transcription unit: RNA polymerase, DNA & growing RNA strand Fig. 17-UN1 Transcription unit Promoter 5 3 3 5 RNA polymerase RNA transcript 3 5 Template strand of DNA Initiation Transcription factors bind first to the promoter in Eukaryotes RNA pol II binds DNA Transcription Initiation Complex is formed Starts to transcribe 5′ 3′ DNA Promoter Nontemplate strand T A T AAAA AT AT T T T TATA box Transcription factors 3′ 5′ 1 A eukaryotic promoter 3′ 5′ 2 Several transcription factors bind to DNA. Start point Template strand 5′ 3′ RNA polymerase II Transcription factors 5′ 3′ 5′ 3′ RNA transcript Transcription initiation complex 3′ 5′ 3 Transcription initiation complex forms. Elongation RNA polymerase moves along DNA Untwists DNA Adds nucleotides to 3’ end Fig. 17-7b Nontemplate strand of DNA Elongation RNA polymerase 3 RNA nucleotides 3 end 5 5 Direction of transcription (“downstream”) Newly made RNA Template strand of DNA Termination Prokaryotes Stop signal Sequence on DNA RNA transcript signals polymerase to detach from DNA RNA strand separates from the DNA Termination Eurkaryotes Polyadenylation signal sequence on mRNA AAUAAA Recognized by RNA polymerase II mRNA is released Transcription Promoter 5′ 3′ RNA polymerase 1 Initiation Transcription unit 3′ 5′ Start point 3′ 5′ 5′ 3′ Template strand of RNA Unwound transcriptDNA DNA 2 Elongation Rewound DNA 3′ 5′ 3′ 3 Termination 3′ 5′ RNA transcript 5′ Direction of transcription (“downstrea m”) 3′ 5′ 5′ 3′ 5′ Completed RNA transcript 3′ Eukaryotes mRNA is modified Nucleus RNA processing Eukaryotes 5’ cap Addition of a GTP 5’ phosphate of the first base of mRNA Methyl group is added to the GTP 3’poly-A-tail Several A’s on the end of the mRNA Eukaryotes Introns: non-coding sequences of nucleic acids Exons: coding sequences of nucleic acids Euraryotes RNA splicing Cut out introns Reconnect exons snRNP’s (small nuclear RNA’s) Spliceosome: Many snRNP’s come together & remove introns Translation Passing the code to make a polypeptide mRNA rRNA ribosomes tRNA Translation Ribosome Located in the cytoplasm Site of translation 2 subunits composed of protein & RNA Small (20 proteins and 1 RNA) Large (30 proteins and 2 RNA) 3 sites on ribosome surface involved in protein synthesis E, P, and A sites Ribosome P site (Peptidyl-tRNA binding site) Exit tunnel A site (AminoacyltRNA binding site) E site (Exit site) E P A mRNA binding site (b) Schematic model showing binding sites Large subunit Small subunit Ribosome Ribsome Growing polypeptide Amino end Next amino acid to be added to polypeptide chain E tRNA mRNA 5′ 3′ Codons (c) Schematic model with mRNA and tRNA Translation tRNA Aminoacyl-t-RNA synthetases Activating enzymes Link correct tRNA code to correct aa One for each 20 amino acids Some read one code, some read several codes 45 tRNA’s Translation Nonsense codes UAA, UAG, UGA code to stop AUG codes for start as well as methionine Ribosome starts at the first AUG it comes across in the code Translation mRNA binds to rRNA on the ribosome mRNA attaches so only one codon is exposed at a time tRNA (anti-codon) Complementary sequence Binds to mRNA tRNA carries a specific amino acid Adds to growing polypeptide Translation 1. Initiation 2. Elongation 3. Termination Initiation Initiation complex 1. tRNA with methionine attached binds to a small ribosome 2. binds at the 5’ cap (Eukayotes) 3. tRNA is positioned on to the mRNA at AUG 4. Initiation factors position the tRNA on the P site 5. Attachment of large ribosomal unit Initiation Requires energy GTP Forms the Initiation complex Initiation 3′ U A C 5′ 5′ A U G 3′ P site Pi + GTP GDP Initiator tRNA E mRNA 5′ Start codon 3′ Small ribosomal subunit mRNA binding site 1 Small ribosomal subunit binds to mRNA. Large ribosomal subunit 5′ A 3′ Translation initiation complex 2 Large ribosomal subunit completes the initiation complex. Elongation Elongation factors Help second tRNA bind to the A-site Two amino acids bind (peptide bond) Translocation: Ribosome moves 3 more nucleotides along mRNA in the 5’to 3’ direction Elongation Initial tRNA moves to E site Released New tRNA moves into A site Continues to add more aa to form the polypeptide Elongation Amino end of polypeptide 1 Codon recognition 3′ mRNA E Ribosome ready for next aminoacyl tRNA P A site site 5′ GTP GDP + P i E E PA PA GDP + P i 3 Translocation 2 Peptide bond formation GTP E PA Termination Release factors: Proteins that release newly made polypeptides Codon (UAG, UAA, UGA) Release factor binds to the codon Polypeptide chain is released from A site Termination Release factor Free polypeptid e 3′ 3′ 5′ Stop codon (UAG, UAA, or UGA) 1 Ribosome reaches a stop codon on mRNA. 5′ 2 Release factor promotes hydrolysis. 5′ 3′ 2 GTP 2 + 2 Pi GDP 3 Ribosomal subunits and other components dissociate. Fig. 17-UN3 mRNA Ribosome Polypeptide Translation <> Growing polypeptides Completed polypeptide Incoming ribosomal subunits Start of mRNA (5′ end) End of mRNA (3′ end) (a) Several ribosomes simultaneously translating one mRNA molecule 2 1 3 Polypeptide SRP SRP synthesis binds binds to begins. to receptor signal protein. peptide . Ribosome mRNA Signal peptide ER LUMEN 5 SignalSRP cleaving detaches enzyme and polypeptide cuts off signal synthesis peptide. resumes. Signal peptide removed SRP CYTOSOL 4 SRP receptor protein Translocation complex 6 Completed polypeptide folds into final conformation. ER membrane Protein Similarities DNA RNA Transcription Protein Translation Differences in gene expression Transcription 1. Prokaryotes one RNA polymerase Eukaryotes 3 RNA polymerases (poli-II mRNA synthesis) 2. Prokaryotes mRNA contain transcripts of several genes Eukaryotes only one gene 3. Prokaryotes no nucleus so start translation before transcription is done Differences in gene expression 3. Eukaryotes complete transcription before leaving the nucleus 4. Eukaryotes modify RNA Introns/exons 5. Prokaryotes Polymerase binds promoters Eukaryotes transcription factors bind first then enzyme 6. Termination Differences in gene expression Translation 1. Prokaryotes start translation with AUG Eukaryotes 5’cap initiates translation 2. Prokaryotes smaller ribosomes Nuclear envelope TRANSCRIPTION DNA PreRNA PROCESSING mRNA NUCLEUS TRANSCRIPTION CYTOPLASM DNA mRNA Ribosome CYTOPLASM TRANSLATION TRANSLATION Ribosom e Polypeptide Polypeptide (a) Bacterial cell mRNA (b) Eukaryotic cell Mutations Changes in genetic information Point mutations: Change in a single base pair Sickle cell mutation Point mutation Wild-type β-globin Wild-type β-globin 3′ DNA C T C G A G 5′ 5′ mRNA G A G Normal hemoglobin Glu Sickle-cell βglobin 5′ 3′ 3′ 3′ 5′ 5′ Mutant β-globin DNA C A C G T G 5′ 3′ G U G 3′ mRNA Sickle-cell hemoglobin Val Mutations Two types 1. Base-pair substitution 2. Insertion or deletion Mutations 1. Base-pair substitution Exchange one nucleotide and base pair with another A. Silent mutations No effect on proteins Silent mutaton Wild type DNA template 3′ T A C T T C 5′ A T G A A G strand mRNA 5′ A U G A A G Protein Met Lys Amino end Nucleotide-pair substitution: silent A A A C C G A T T 5′ T T T G G C T A A 3′ U U U G G C U A A 3′ Phe Gly Stop Carboxyl end A instead of AGC C A A T T 5′ 3′ T A C T T C A A 5′ A T G A A G T T T G G T T A A 3′ 5′ A U G A A G U U Met Lys U instead of UCG G U U A A 3′ Phe Gly Stop Mutations B. Missense mutations: Substitutions that change one aa for another Little effect Missense Wild type DNA template 3′ T A C T T C 5′ A T G A A G strand mRNA 5′ A U G A A G Protein Met Lys Amino end Nucleotide-pair substitution: missense A A A C C G A T T 5′ T T T G G C T A A 3′ U U U G G C U A A 3′ Phe Gly Stop Carboxyl end T instead of A CA A T C G A T T 5′ 3′ T A C T T C 5′ A T G A A G T T T A G C T A A 3′ 5′ A U G A A G Met Lys A instead of UGU U A G C U Phe Ser A A 3′ Stop Mutations C. Nonsense mutations Point mutation codes for stop codon Stops translation too soon Shortens protein Non-functional proteins Mutations 2. Insertions or deletions Additions or losses of nucleotides Frameshift mutations Improperly grouped codons Nonfuctional proteins Fig. 17-23 Wild-type DNA template strand 3 5 5 3 mRNA 5 3 Protein Stop Amino end Carboxyl end A instead of G 3 5 Extra A 5 3 U instead of C 5 5 3 3 5 Extra U 3 5 3 Stop Stop Silent (no effect on amino acid sequence) Frameshift causing immediate nonsense (1 base-pair insertion) T instead of C missing 3 5 5 3 3 5 5 3 A instead of G missing 3 5 5 3 Stop Missense Frameshift causing extensive missense (1 base-pair deletion) missing A instead of T 3 5 5 3 3 5 3 5 U instead of A 5 5 3 missing Stop Stop Nonsense (a) Base-pair substitution 3 No frameshift, but one amino acid missing (3 base-pair deletion) (b) Base-pair insertion or deletion Mutagens Chemical or physical agents Mutations in DNA