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Chapter 17 PROTEIN SYNTHESIS: FROM GENE TO PROTEIN

Chapter 17 PROTEIN SYNTHESIS: FROM GENE TO PROTEIN. Central Dogma of Biology. Flow of genetic information:. RNA (ribonucleic acid). RNA has many functions in the cell. 1. pre-mRNA : precursor to mRNA, newly transcribed and not edited

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Chapter 17 PROTEIN SYNTHESIS: FROM GENE TO PROTEIN

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  1. Chapter 17PROTEIN SYNTHESIS: FROM GENE TO PROTEIN

  2. Central Dogma of Biology • Flow of genetic information:

  3. RNA (ribonucleic acid)

  4. RNA has many functions in the cell. • 1.pre-mRNA: precursor to mRNA, newly transcribed and not edited • 2. mRNA: carries the code from DNA that specifies amino acids • 3. tRNA: carries a specific amino acid (anticodon)to ribosome • 4. rRNA: makes up 60% of the ribosome; site of protein synthesis • 5. snRNA: small nuclear RNA; part of a spliceosome (in RNA splicing) • 6. srpRNA: a signal recognition particle that binds to signal peptides • 7. RNAi: interference RNA; a regulatory molecule • 8. ribozyme: RNA molecule that functions as an enzyme

  5. 1. messengerRNA (mRNA) single uncoiled long strand - transmits DNA info during protein synthesis - serves as template to assemble amino acids transfer RNA (t RNA) - carries amino acids to ribosome ribosomal RNA (r RNA) makes up large part (2/3) of ribosome - globular 3 types RNA directly involved in making proteins

  6. PROTEIN SYNTHESIS/GENE EXPRESSION • Formation of proteins using information coded on DNA and carried out by RNA. • one gene = one RNA molecule • Gene: region of DNA whose final product is • either a polypeptide or RNA molecule • A gene is expressed when protein synthesis is occurring.

  7. The Genetic Code • How is information necessary for creating proteins encoded in the RNA? • The genetic code from DNA is transcribed onto m RNA by Codons. • For each gene, one DNA strand is the template strand • mRNA (5’ 3’) complementary • to template • mRNA triplets (codons) code for • amino acids in polypeptide chain

  8. Code word/Codon(triplet): • specific group of 3 successive bases on DNA and mRNA • - codes for a specific amino acid to be placed on the protein chain • - 20 biological amino acids, but more than 20 codons • Like “genetic words” • DNA code words: ACT, GCA, TTA • RNA codons: UGA, CGU, AAU

  9. How many combinations of code words can we make from 4 bases? • 64 different codon combinations ( 43 = 64) • ** each code word always codes for same amino acid** • Redundancy: 1+ codons code for each of 20 AAs • Reading frame: groups of 3 must be read in correct groupings • This code is universal: all life forms use the same code

  10. It is possible for non-Watson-Crick base pairing to occur at the third codon position. • This has phenomenon been termed the • wobble hypothesis.

  11. How do these code words affect protein synthesis? • Order of code words • codes for • Order of amino acids • codes for • Specific type of protein

  12. BUILDING OF PROTEINS • - DNA unzips • - original strand of DNA acts as template for m RNA • (only one strand of DNA molecule needed for transcription) • euchromatin: uncoiled areas of DNA, active site of transcription • antisense /non-template strand:non coding strand of DNA • sense/template strand: coding strand of DNA

  13. 2 Stages of Protein Synthesis

  14. Stages of Protein Synthesis • Transcription (nucleus) • Process where mRNA is produced from DNA • Transcription unit: stretch of DNA that codes for a polypeptide or RNA

  15. Transcription • Initiation (prokaryotes) • RNA polymerase binds • directly to promoter in • DNA • - promoter : region of DNA that initiates transcription of a particular gene. • - located near the transcription start sites of genes on sense strand • - located upstream on the DNA (towards the 5’ region of the antisense strand) • **can be about 100–1000 base pairs long** nontemplate strand template strand

  16. Steps of transcription Initiation (eukaryotes) A. The promoter site on the DNA contains a sequence called a TATA box - TATAAAA - recognized by RNA polymerase ll - can be up to 25 bases upstream from point of transcription B. Transcription factor proteins and RNA polymerase ll bind to promoter section of DNA and unwinds the part of the DNA to be transcribed. (this is the structural gene: codes for a single protein)

  17. 2. Elongation A. RNA polymerase reads DNA template strand B. Complementary nucleotides are added to the 3' end of RNA using information in DNA as instructions **Polymerases always work from the 3' to the 5' end of the coding strand of DNA (template); thus the antiparallel structure it is forming is going from the 5' to 3’ direction. C. As RNA polymerase moves, it untwists DNA, then rewinds it after mRNA is made D. Once RNA nucleotides are attached to DNA chain, codons are in proper order

  18. Termination • RNA polymerase transcribes a • terminatorsequence in DNA • mRNA and polymerase detach. • * prokaryotes- mRNA ready • to use • * eukaryotes- pre-mRNA, • will undergo further • modifications • transcription animation

  19. RNA Processing Occurs After Transcription • 1. Alteration of pre-mRNA ends • - 5’ cap (modified guanine) and 3’poly-A tail(50-520 A’s)are added • - functions: • help export mature mRNA from nucleus • protect mRNA from enzyme degradation • help ribosomes attach to mRNA

  20. RNA Processing Occurs After Transcription • RNA Splicing • - Pre-mRNA has introns (noncoding sequences) andexons (codes for amino acids) • - Splicing- introns cut out, exons joined together • - Once RNA is spliced it can move out of the nucleus to be translated

  21. RNA Splicing, cont. • snRNPs • small nuclear ribonucleoproteins • snRNP = snRNA + protein • Pronounced “snurps” • Recognize splice sites • snRNPs join with other proteins to form a spliceosome Spliceosomescatalyze the process of removing introns and joining exons Ribozyme = RNA acts as enzyme • RNA splicing animation

  22. Importance of Introns • Functions not known for most introns • Some regulate gene expression • Alternative RNA Splicing: produce different combinations of exons • Depends which segments are treated as exons during splicing • One gene can make more than one polypeptide • 20,000 genes  100,000 polypeptides • Changes in gene expression may confer an evolutionary advantage

  23. Prokaryotic vs Eukaryotic Gene Expression • Prokaryotes • Transcription and translation both in cytoplasm • RNA poly binds directly to promoter • No introns • No RNA splicing • Eukaryotes • Transcription in nucleus; translation in cytoplasm • DNA in nucleus, RNA travels in/out nucleus • RNA poly binds to TATA box & transcription factors • RNA contains introns • RNA splicing

  24. Translation (in cytoplasm at ribosome) • - process whereby protein is synthesized from mRNA • - newly synthesized mRNA moves from nucleus to ribosome in cytoplasm • - gene has 3x more nucleotides than the protein it makes • Ex: 100 a.a. = 300 nucleotides • - components of translation • mRNA- message • tRNA- interpreter • ribosome- site of translation

  25. Transfer RNA (t RNA) - function: transfers amino acids to ribosome - 20 types – one for each amino acid (specific) - structure (cloverleaf) - found in cytosol Aminoacyl-tRNA-synthetase: enzyme that binds tRNA to specific amino acid

  26. Ribosomes • - made in nucleolus • 2 subunits make up ribosome • - about 2/3 is r-RNA and 1/3 is protein • - smaller subunit has binding site for mRNA • - normally apart in cytoplasm, come together during protein synthesis

  27. Steps of translation • 1. Initiation • A. 5’ end of m RNA binds to small subunit • B. initiator tRNA carrying Met attaches to P site • C. large subunit attaches (ribosome ready for protein synthesis) • - sites: locations on ribosome where tRNA anticodons attach • P (peptidyl) site- holds aa chain A (aminoacyl) site- holds aa to be added E (exit) site- exit for tRNA • * start codon (AUG) will be at the site • on mRNA where this occurs • ** anticodon on first tRNA will always be UAC, amino acid 1 will always be methionine

  28. 2. Elongation A. t-RNA with a specific anticodon binds a specific amino acid. This happens for several t-RNAs and proper corresponding amino acids in the cytoplasm. ATP: energy source used to bind the amino acid to the t-RNA. Aminoacyl-tRNA synthase: enzyme that does the binding. B. First tRNA binds to P site, second tRNA binds to A site (anticodons are complementary to mRNA codons) C. Peptidyl transferase reaction occurs: #1 a.a. joins to #2 a.a. D. Ribosome moves down mRNA and first tRNA is released to be used over again - translocation: movement of ribosome down mRNA E. Amino acids continue to be added to protein chain thru same mechanism - peptidyl synthase: enzyme that joins a.a. together

  29. Termination • A. stop codon is reached (UAA, UGA, or UAG). • B. release factor binds to stop codon and polypeptide is released • C. subunits dissociate (can be used over again) • D. protein is released into cell • E. mRNA is broken down by cell (not be used again – only once) • F. tRNA is released into cell (used over again)

  30. Speed of Translation • - process occurs from minutes to hours in an organism • Polyribosomes: strings of ribosomes that can translate many copies of a polypeptide very quickly • During synthesis, polypeptide chain coils and folds spontaneously • Protein synthesis animationProtein synthesis animation 2

  31. Where to proteins go once synthesized? • Free ribosomes(floating in cytosol) • make proteins that stay in cytosol to perform functions • Bound ribosomes(attached to ER) • make proteins for secretion • Use signal peptide to target their location • make proteins of the endomembrane system • nuclear envelope, ER, Golgi, lysosomes, vacuoles, plasma membrane)

  32. Signal Mechanism • Signal peptide: 20 AA at leading end of polypeptide determines destination • Signal-recognition particle (SRP): brings ribosome to ER

  33. Mutations • Changes in genetic code of a cell • Source of new genes and diversity of genes among organisms • Mutagen: substance that causes mutations • Radiation, chemicals, viruses • Large scale (chromosome) • Involve large segments of chromosome • Cause disorders or death • Types: duplications, large deletions, translocation, inversion, • non-disjunction • Small scale • Single nucleotide-pair substitutions • Nucleotide-pair insertions or deletions (one or more pairs)

  34. Large Scale Chromosome Mutations Review

  35. Small Scale Mutations • Point mutations: alter 1 base pair of a gene • Base-pair substitutions– replace 1 with another • Silent: no change in amino acid due to redundancy • Missense: change one amino acid into another • Nonsense: change into stop codon, results in shorter non-functional polypeptide • Frameshift: alters reading frame of RNA • causes non-functional proteins • Insertions: addition of nucleotide/s • Deletions: removal of nucleotide/s

  36. Base Pair Substitutions • Substitution: Silent (no effect)

  37. Base Pair Substitutions • Substitution- Missense (change of one amino acid to another)

  38. Sickle Cell Anemia: point mutation

  39. Base Pair Substitutions • Substitution: Nonsense • (change into stop codon)

  40. Frameshift • Insertion: shifts frame to right

  41. Frameshift • Deletion: shifts frame to left, premature termination

  42. Summary of Protein Synthesis

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