Bio 99 Final Lec 17-18

  1. What you need to know about proteins
    • Proteins are chains of amino acids joined together
    • There are 20 different amino acids (A.A.)
  2. Features of proteins:
    • Amino group – has N
    • Carboxyl group – has C
    • R group – “side chain”, each amino acid has a different R
  3. The bond that joins two amino acids together is called a ____ ____
    peptide bond
  4. Proteins are also called ____
  5. N-terminus
    front of polypeptide, similar to 5’ on DNA/RNA
  6. C-terminus
    back end of polypeptide, similar to 3’ on DNA/RNA
  7. tRNA structure
    • Each tRNA has 4 to 5 “arms”
    • Anticodon arm – contains the anticodon, interacts with mRNA sequence
    • Amino acid arm – attaches to amino acid 
    • Other arms – structural, interact with ribosome, tRNA synthetase
  8. tRNATyr
    tRNA that recognizes a codon for Tyrosine, but does not necessarily have a Tyrosine amino acid attached to it.
  9. Tyr-tRNATyr
    tRNA that recognizes a codon for Tyrosine, and is “charged” with a Tyrosine amino acid.
  10. Aminoacyl-tRNA
    a tRNA with an amino acid attached to it.
  11. Base modification
    specific nucleotides are modified
  12. Cleavage
    the ends of the transcript are removed
  13. CCA addition
    a CCA is attached to the 3’ end of the transcript. This is what the amino acid attaches to
  14. Introns removed only in....
  15. At which location, A, B, C or D, will the
    anticodon be found?

    Image Upload 1
    D. )
  16. At which location, A, B, C or D, will the
    CCA attached?

    Image Upload 2
    D. )
  17. tRNA Activation
    • Each tRNA must have its correct amino acid attached to it
    • 2 steps: 1) Adenylylation and 2) tRNA charging
    • Both steps catalyzed by aminoacyl-tRNA synthetases (AATS), within the same active site
    • There are 20 different aminoacyl-tRNA synthetases, 1 for each amino acid
    • There are more than 20 tRNAs though, so each AATS can recognize more than one tRNA
  18. tRNA Activation, Step 1: Adenylylation
    • Amino acid + ATP converted into aminoacyl-AMP + PPi
    • AMP attaches to carboxyl group of amino acid
    • Pyrophosphate (PPi) is generated in this reaction, which is later hydrolyzed into 2 phosphates, providing the energy to drive this reaction as well as make it irreversible
  19. tRNA Activation, Step 2: tRNA charging
    • aminoacyl-AMP + tRNA into aminoacyl-tRNA + AMP
    • Aminoacyl transferred off of AMP onto the tRNA’s CAA arm
  20. 2 classes of aminoacyl-tRNA
    synthetases do this differently
    • Class I - attach amino acid onto 2’OH of CCA
    • Class II – attach amino acid onto 3’OH of CCA
  21. The addition of the charged amino acid to the
    3’ adenine of a tRNA is being carried out by
    a. Class I aminoacyl-tRNA synthetase.
    b. Class II aminoacyl-tRNA synthetase.
  22. Necessary
    Something is necessary for a function when you need it to carry out that function
  23. How to test if item A is necessary for a function
    Remove item A and see if function is retained
  24. Sufficient
    Something is sufficient for a function when you can get function with only that thing
  25. How to test if item A is sufficient for a function
    Add item A in isolation (where you only have item A) and see if function can occur
  26. You identify another motif (motif K) which contains a helix-turn-helix motif,
    and you’re very sure it is the DNA binding motif for protein X. Which
    strategy would best show that the motif is sufficient for DNA binding?
    A. Create a fusion protein with motif K, plus the transactivation domain of
    another protein and see if the fusion protein activates transcription of a
    plasmid that contains the regulatory site for protein X
    B. Create a nonsense mutation in the motif and see if it can still bind DNA
    C. Delete that motif from the gene and see if the mutant protein can bind
    to DNA
    D. Create a mutant protein X that cannot bind DNA, then try to create a
    reversion mutation to restore DNA binding. See if the mutation and
    reversion mutation occur within the motif
  27. Aminoacyl-tRNA synthetases can distinguish between tRNAs
    • The identity nucleotides are the
    • nucleotides of a tRNA that are recognized by their specific synthetases (orange circles).
    • Identity nucleotides are scattered throughout the tRNA
    • Each synthetase recognizes a unique set of identity nucleotides
    • Two different tRNAs for the same amino acid will share the same identity nucleotides, which allows them to be recognized by the same synthetase.
  28. AA-tRNA proofreading
    Case 1 – A larger AA trying to attach to a smaller AA’s tRNA
    • Amino acid binding site
    • If AA too big – doesn’t fit
    • If AA is too small - fits
  29. AA-tRNA proofreading
    Case 2 – A small AA trying to attach to a larger AA’s tRNA
    Ile-tRNA synthetase has an acylation site, as well as a separate proofreading site
  30. Bacteria:
    Initiator Met tRNA
  31. Bacteria
    Internal Met tRNA
  32. ________ converts Methionine to N-formylmethionine (fMet)
  33. fMet
    • Formyl group attaches to N-terminus of fMet, preventing fMet from being able to attach to an AA in front of it. fMet can only be the first AA.
    • Cannot be added internally
    • tRNAfMet is the only tRNA that is recognized by the ribosome initiation complex
  34. Eukaryotes:
    Initiator tRNA
  35. Eukaryotes:
    Internal tRNA
  36. In both bacteria and eukaryotes, ______ often remove the Nterminal Met, so many mature proteins don’t have Met as the first AA.
  37. Ribosomes contain 2 subunits (Bacteria)
    • Small subunit - 30S
    • rRNA – 16S
    • Protein – 21 total subunits (S1 to S21)
    • Function – mRNA, tRNA assembly
    • Large subunit – 50S
    • rRNA – 5S and 23S
    • Protein – 36 total subunits (L1 to L36)
    • Function – catalyze peptide bond formation
  38. Ribosomes contain 2 subunits (Eukaryotes)
    • Eukaryotes
    • Similar to bacteria but slightly
    • more complex, more proteins
    • 40S small and 60S large
  39. Puromycin
    • mimics tRNA, but binds directly to the large subunit, ribosomes will attach it to a growing polypeptide chain
    • New amino acids cannot be attached to puromycin, so it terminates translation and is therefore a powerful antibiotic
    • In this experiment, puromycin is just the substrate for forming peptide bonds
  40. Ribozyme
    RNA with enzymatic activity
  41. The ribosome has 3 tRNA binding sites (name them)
    • A site
    • P site
    • E site
  42. A site
    – Acceptor site – where new tRNAs enter ribosome
  43. P site
    – Polypeptide site – where growing polypeptide chain is held
  44. E site
    – Exit site – where tRNAs are expelled after amino acid removal
  45. Which ribosomal subunit holds the mRNA?

    D. )
  46. Which ribosomal subunit catalyzes peptide bond formation?

    D. )
  47. Shine-Dalgarno sequence
    • consensus sequence in front of start codon
    • Recruits small subunit to mRNA
    • Directs mRNA start site to correct position on ribosome
    • Base pairs with 16S rRNA on small subunit
  48. Bacterial proteins involved in initiation
    • IF-1 – Fills A site to prevent tRNAs from binding
    • IF-2 – escorts initiator tRNA
    • IF-3 – prevents large subunit from binding
  49. Translation Initiation in Bacteria, Step 1a-blocking
    • IF-1 and IF-3 bind to 30S small
    • subunit
    • IF-1 – blocks A site, prevents tRNA binding
    • IF-3 – blocks large subunit from binding
  50. Translation Initiation in Bacteria, Step 1b- mRNA recruitment
    • mRNA attaches to 30S small subunit
    • uses Shine-Dalgarno sequence to position start codon right at P site
  51. Translation Initiation in Bacteria, Step 2 recruitment of initiator tRNA
    • IF-2 binds to initiator tRNA
    • IF-2 is bound to GTP
    • Has GTP-hydrolase activity

    • Initiator tRNA (fMet-tRNAfMet) binds to start codon
    • rRNA binds to unique sequence on initiator tRNA (reason why internal tRNAMet doesn’t bind)
    • tRNAfMet can only bind to the P site
    • No other tRNAs can bind to P site
  52. Translation Initiation in Bacteria,  
    Step 3 – recruitment of large subunit
    • 30S changes conformation to kick out IF-3, allowing 50S large subunit to bind
    • Hydrolysis of GTP to GDP causes IF-1 and IF-2 to leave

    • Initiation complex completed
    • Both large and small subunits bound
    • mRNA with start codon lined up
    • Initiator tRNA bound to start site in P site of ribosome
  53. What is/are the function(s) of the Shine-Dalgarno sequence?

    E. )
  54. Which factor is responsible for preventing tRNAs from prematurely base
    pairing with the mRNA codons?

    D. )
  55. What does the hydrolysis of GTP to GDP help to facilitate?

    D. )
  56. Initiation in Eukaryotes, Step 1– blocking sites on small subunit
    • eIF1A binds to and blocks A site on small subunit to prevent tRNA binding
    • eIF3 (and eIF1) block large subunit from assembling
  57. Initiation in Eukaryotes, Step 2 – loading initiator tRNA
    • eIF2 binds to initiator tRNA (Met-tRNAi Met) and escorts into P site of small subunit.
    • eIF2 is also bound to GTP
    • unique sequences on initiator tRNA allow eIF2 to bind
  58. Initiation in Eukaryotes, Step 3 - loading the mRNA
    • eIF4F – complex that binds to 5’ cap of mRNA and escorts it to small subunit. (no real bacterial equivalent, but partly serves similar role as Shine-Dalgarno sequence)
    • Contains 3 factors
    • 1) eIF4E – Binds to 5’ cap
    • 2) eIF4A – ATPase and RNA helicase
    • 3) eIF4G – Adapter, binds to eIF3 and eIF4E, linking mRNA to small subunit
    • Binding of eIF4F/mRNA to pre-initiation complex requires hydrolysis of ATP (by eIF4A)
    • Unlike bacteria, mRNA binds at 5’ cap (not start codon)
  59. Initiation in Eukaryotes, Step 4 – Scanning for the start codon
    • Scanning – complex travels along mRNA until first start codon is found, then stops
    • Kozak sequence helps identify start codon (similar to Shine-Dalgarno)
    • Scanning implies that translation usually starts with the first AUG from the 5’ cap
  60. Initiation in Eukaryotes, Step 5 – Loading large subunit
    • Both GTPs (bound to eIF2 and eIF5B) are hydrolyzed This drives the release of all initiation factors
    • Unlike bacteria, 2 GTPs are required (in bacteria, only 1)
    • Plus, 1 ATP was required to load mRNA
    • eIF4F and 5’ cap of mRNA also released
    • Large subunit can now bind to pre initiation complex and assembly of the initiation complex is now complete
  61. Like the Shine-Dalgarno sequence in bacteria, the eukaryotic Kozak
    sequence helps the small subunit to find the start codon. What other
    eukaryotic factor helps to fulfill a different part of the function of the
    Shine-Dalgarno sequence?

    D. )
  62. Which factor is not bound to GTP or ATP, or involved in its hydrolysis?

    A. )
  63. What is a feature of eukaryotic translation initiation that is different from bacteria?

    A. )
  64. Bacterial Polysomes
    • Multiple ribosomes can translate the same mRNA simultaneously
    • In bacteria, this can even happen while the mRNA is still being transcribed
    • Multiple RNA polymerases can also be transcribing the same DNA at once
  65. Eukaryote Polysomes
    • eIF4G (part of eIF4F complex) can bind to poly-A binding protein (PABP)
    • Connects 5’ cap to poly-A tail, forming a circle
    • Facilitates translational regulation
  66. Internal Ribosomal Entry Site (IRES)
    • Most of eukaryotic translation is called cap-dependent translation, but translation can also happen without using the 5’ cap and first start codon
    • Internal ribosomal entry site (IRES) – sequence that can bind eIF4F and direct ribosome assembly and translation away from the 5’ cap
    • Used by viruses that block cap dependent translation, but allows their own genes to translate
    • Viruses cleave eIF4G to block its ability to tether mRNA to small subunit
    • Small eIF4G fragment sufficient to direct IRES to ribosome
    • Some eukaryotic genes also use IRES
    • IRES is also used by molecular biologists to express two genes off the same transcript
    • Essentially makes eukaryotic polycistronic transcripts
  67. What does eIF4G do?
    A. Tethers the mRNA to the small subunit
    B. Tethers the 5’ cap of mRNA to the poly (A) tail
    C. Hydrolyzes GTP to separate eIF4F from the small subunit
    D. Hydrolyzes ATP to bind the mRNA to the small subunit
    E. A and B
    A and B
  68. A virus shuts down cap-dependent translation, which of these happen next?

    D. )
  69. What is the function of the 48S particle?

    B. )
Card Set
Bio 99 Final Lec 17-18