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From DNA to Protein. Chapters 10 & 11. Overview. Review of DNA & RNA Transcription & Translation Gene Mutations Controls over Genes. DNA: A Review. Holds: Genetic information Protein-building instructions. Double-helix of nucleotide bases with sugar-phosphate backbone

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    1. From DNA to Protein ... Chapters 10 & 11

    2. Overview • Review of DNA & RNA • Transcription & Translation • Gene Mutations • Controls over Genes

    3. DNA: A Review Holds: Genetic information Protein-building instructions

    4. Double-helix of nucleotide bases with sugar-phosphate backbone Bases held together by H-bonds: • A always pairs with T • G always pairs with C

    5. So what is a gene? Segment of DNA molecule Carries instructions for 1 polypeptide chain Bases grouped in triplets that code for specific amino acid Variations in arrangement of bases lets cells make all proteins needed

    6. Exons = protein-coding base sequences Introns = non-coding, repetitive sequences (genome scrapyard of ready-to-use DNA segments & small RNA molecules) Both transcribed but introns removed before mRNA reaches cytoplasm

    7. RNA: A Review Similar to DNA, except: • Single-stranded • Uracil replaces thymine • Adenine pairs with uracil Decodes DNA & acts as messenger

    8. Types of RNA: mRNA Messenger RNA Carries protein-building instructions from gene to ribosome “Half-DNA”

    9. Types of RNA: rRNA Ribosomal RNA One of components of ribosomes With tRNA, translate protein-building instructions carried by mRNA

    10. Ribosomes 2 subunits of rRNA & structural proteins Have 2 tRNA binding sites Come together as whole functional ribosome during translation

    11. Ribosomes of prokaryotes and eukaryotes are similar in function but different in composition Certain antibiotics (e.g. tetracycline, streptomycin) inactivate prokaryotic ribosomes but don’t affect eukaryotic ribosomes

    12. Types of RNA: tRNA Transfer RNA 45 different types With rRNA, translate protein-building instructions carried by mRNA

    13. Has anti-codon head: = 3-base sequence complementary to codon on mRNA transcript Anti-codon head is complementary to amino acid it carries

    14. 45 tRNAs exist in eukaryotic cells Codon-anticodon pairing has “wiggle room” for 3rd base of codon e.g. AUU, AUC, AUA (isoleucine) use same tRNA

    15. The Genetic Code The rules that link codons in RNA with the corresponding amino acids in proteins Bases read 3 at a time = codon 64 codons that code for 20 amino acids Some amino acids have ≥ 1 codon (↓ transcription & translation errors) AUG = methionine = START UAA, UAG, UGA = STOP

    16. Transcription & Translation Process that turns sequence of nucleotide bases in genes into sequence of amino acids in proteins transcription translation DNA RNA protein

    17. DNA base sequence acts as template to make RNA Occurs in eukaryotic nucleus RNA moves into cytoplasm Amino acids join to become polypeptides (proteins)

    18. Transcription DNA gene’s base sequence → complementary mRNA base sequence First step in protein synthesis Sequence of nucleotides bases on DNA strand exposed Becomes template for RNA to be built from A, C, G, T

    19. Transcription factor binds to promoter (START) base sequence on DNA Promoter determines where mRNA synthesis begins & which DNA strand is template

    20. RNA polymerase binds to promoter (unwinds 16-18 bps of DNA helix) RNA polymerase moves along protein-coding gene region RNA polymerase unwinds DNA in front & rewinds behind as mRNA elongates

    21. Incoming RNA nucleotides bind with complementary bases on template strand e.g. (AGC) on DNA → (UCG) on mRNA Creates complementary sequence from DNA base sequence  template mRNA is released at end of gene region (STOP)

    22. Is actually pre-mRNA because has intron junk mRNA modified before leaving nucleus = introns cut out & exons respliced to form functional mRNA mRNA associates with proteins & leaves nucleus = is now ready for protein synthesis

    23. mRNA enters cytoplasm = location of pool of tRNA & free amino acids Protein synthesis (translation) begins

    24. Translation mRNA base sequence → amino acids → proteins mRNA transcript enters ribosome Codons translated into polypeptide chain

    25. Initiation of Translation mRNA binds to small ribosomal unit Initiator tRNA binds to start codon (AUG) (this tRNA carries Met & has anti-codon UAC) Large ribosomal subunit binds to small subunit to form functional ribosome

    26. Initiator tRNA fits into P site of ribosome (P site holds growing polypeptide) A site lies vacant for the next amino-acid-carrying tRNA

    27. Elongation of Translation Chain of polypeptides is synthesized as mRNA passes between ribosomal subunits tRNAs transfers amino acids from cytosol to ribosome Elongation is a 3-step process

    28. 1. Codon recognition: Anti-codon of incoming amino-acid-carrying tRNA pairs with mRNA codon in A site Amino acids bind to mRNA in order dictated by template of codons

    29. 2. Peptide bond formation: Polypeptide separates from tRNA in P site & attaches to amino acid carried by tRNA in A site Peptide bond catalyzed by rRNA in large ribosomal subunit

    30. 3. Translocation: P site tRNA leaves ribosome Ribosome moves tRNA in A site (with attached polypeptide) to P site (mRNA moves along too) Next mRNA codon is brought into A site Elongation begins over again for next addition

    31. Polyribosomes Once mRNA passes through ribosome, may become attached to multiple other ribosomes in row Allows many copies of same protein to be made quickly & simultaneously

    32. Termination of Translation mRNA STOP codon enters ribosome (no tRNA has complementary anticodon) Release factors bind to ribosome & detach mRNA & polypeptide chain Ribosome separates back into 2 subunits Proteins either: • Join pool of free proteins in cytoplasm • Enter RER to be modified for transport

    33. Phe Gly Arg Phe Summary of Transcription & Translation Genetic info → protein synthesis Via info transfer of complementary base pairing

    34. Gene Mutations Most mutations are spontaneous & occur during DNA replication DNA polymerases & ligases (proofreaders) catch most errors but not all Bases can be substituted, inserted, deleted Effects on protein structure & function depend on how mRNA sequence is changed

    35. Point Mutations a.k.a. base substitution Single nucleotide replaced with different nucleotide Can be harmless if still codes for same amino acid Can be harmful or even fatal (wrong amino acid can alter protein function or even code for STOP)

    36. a. Missense mutation Substitution alters codon so that it codes for different amino acid Usually changes protein function (good / bad / neutral effects) GCA-UUC-GUC ala - phe - val GCA-UUA-GUC ala - leu - val

    37. b. Nonsense mutation Substitution alters codon so that it codes for STOP signal Results in premature termination of translation Shortened protein is usually non-functional GCA-UAU-GUC ala - tyr - val GCA-UAG-GUC ala - STOP

    38. c. Silent mutation Substitution occurs in 3rd base of mRNA codon New codon codes for same amino acid (does not affect protein function) GCA-UUC-GUC ala - phe - val GCA-UUU-GUC ala - phe - val

    39. Frameshift Mutations 1 or more base inserted or deleted Deletion or insertion shifts 3-base reading window Protein is generally useless = extensive missense & eventually nonsense

    40. Mutagens Some mutations are not spontaneous Ionizing radiation (e.g. x-rays) = break up chromosomes & deposit free radicals in cells Non-ionizing radiation (e.g. UV radiation) = changes base-pairing properties due to thymine sensitivity

    41. When are mutations good? If occur in somatic (body) cells, only affects individual (not heritable) If occur in gametes (sex cells), may be heritable • Can result in harmful, beneficial, or neutral effects on individual’s survival • Adaptation or elimination?

    42. Cell Differentiation Body cells differ in composition, structure, & function Each cell type undergoes selective gene expression = determines which tissues & organs develop

    43. How Are Genes Regulated? Differentiated cells contain all genes BUT Cells only express genes necessary for their specialized functions

    44. Human genome = 25,000 – 30,000 genes Most transcribed only in certain cells at certain times (default state = off) Some transcribed in all cells because encode proteins / RNA that are essential for life = housekeeping genes

    45. Animal development is directed by cascades of gene expression & cell-to-cell signalling Homeotic gene = master control gene that regulates all other genes

    46. Gene Control How fast & when genes will be transcribed & translated Whether gene products are switched on or silenced = Controls over what kinds & how much of each protein are in a cell