Molecular Bio Exam Questions

  1. How is tRNA a bifunctional molecule?
    • 1) decodes mRNA through direct base-pairing interactions
    • 2) synthesizes polypeptide chain from the amino acid they carry
  2. What enzyme is responsible for attaching the amino acid to the tRNA molecule and the specificity of the tRNA amino acid pair?
    Aminoacyl-tRNA Synthetases

    Process: aminoacetylation (adding a specific amino acid to the tRNA)
  3. How does the tRNA molecule ensure fidelity? (2)
    • Fidelity of decoding depends on...
    • 1) Loading the tRNA with the correct amino acids
    • 2) specific interactions and direct pairing between the mRNA codon and the tRNA anticodon
  4. What is the ribosome responsible for and what makes up the ribosome? (3)
    • Ribosome moves processively along the mRNA (5' to 3') and synthesizes a working protein, mediating interaction between mRNA and tRNA. It is composed of:
    • -Small Subunit: responsible for deciphering mRNA and mediating interaction between mRNA and tRNA
    • -Large Subunit: catalyzes formation of peptide bonds between amino acids

    Composed of 2/3 rRNA and 1/3 r-protein
  5. Translation Factors

    What are majority of them?
    How can they regulate cell function?
    Not permanent components of the ribosome that increase fidelity by associating with the ribosome. When they finish their job, they leave.

    Majority of them are GTPases

    The cell can increase and decrease TF synthesis to regulate protein synthesis.
  6. What 2 processes are essential during translation? What links the 2 processes together?
    • 1) Deciphering the triplet codons in the mRNA
    • 2) incorporating the amino acids to the polypeptide chain

    tRNA's link the 2 processes together because of their structure
  7. Describe the basic structure of a tRNA
    • Image Upload 1 
    • Arpund 74-94 nucleotides with 4 stems are base paired with single stranded regions at the end with a 3D L-shape.
  8. Acceptor Stem
    (tRNA) Where the 5' and 3' ends of the tRNA meet (the double stranded region)
  9. 3' CCA tail
    (tRNA) The attachment point of amino acids that get incorporated into a polypeptide chain.
  10. Anticodon Loop
    How does it ensure fidelity?
    (tRNA) 3 nucleotides that collectively base pair with mRNA codon, providing a physical link between tRNA and mRNA.

    Fidelity - the anticodon loop has a specific confirmation that positions the anticodon in the correct location on the mRNA
  11. What is the benefit of having 10% of the nucleotides on the mRNA modified?
    • -aids in folding
    • -stabilizes the overall structure
    • -increases speceficity
    • -allows for cell regulation
  12. How is the L-shape of the tRNA made possible? What is the benefit of the L-shape?
    The L-shape is made possible due to the stabilization of the nucleotides in the D-loop and T-loop.

    The benefit of the L-shape is appropriately spaces the anticodon and the acceptor stem for synthetase and ribosome.
  13. What is the benefit of having a triplet codon? What do triplet codons specify for?
    Triplet codon increases the amount of nucleotides it can code for.

    • Sense Codon - codes for amino acid
    • Nonsense Codon - codes for no amino acid (like a stop codon)
  14. Isoacceptor
    tRNAs that are loaded with the same amino acid. Typically the first 2 bases are conserved and read with strict Watcon and Crick base pairing by forming my RNA helix.
  15. Wobble Pairing
    What does it do?
    What is the benefit?
    What is the typically wobble pair?
    • Image Upload 2
    • Promiscuity reading of the 3rd codon, allowing non Watson and Crick interactions. The benefit of it is it reduces the number of tRNAs needed to decode mRNA codon. (Typically a G-U base pair)
  16. What is the first step to the AA acid being added to the tRNA?
    AA + tRNA energetically unfavorable...

    Amino acid must be activated through the attachment of AMP to form aminoacyl adenylate. The high energy bond can easily be broken and thus transferred to tRNA.

    • Attachment of AMP releases pyrophosphate that produces 2 phosphates.
    • Image Upload 3
  17. What protects the amino acid on the tRNA from spontaneous hydrolysis?
    EFTu (bacteria) eEF1A (eukaryotes, archaea) bind to the aminoacyl tRNA
  18. How would you write a tRNA with a specific amino acid attached to it?
  19. How does the synthetase recognize the correct tRNA?
    Due to tRNA specific structure and sequence known as identity elements in anticodon loop and acceptor stem
  20. What 4 characteristics of the amino acid does the synthetase use to differentiate between amino acids?
    • -charge
    • -hydrophobicity
    • -size
    • -shape
  21. How does an aminoacyl-tRNA synthetase exclude large AA's?
    A large amino acid cannot fit into the active site of the aminoacyl-tRNA synthetase.
  22. How does an aminoacyl-tRNA synthetase prevent smaller amino acids from being incorporated to tRNA (pre-transfer)?
    An aminoacyl-tRNA synthetase has an editing site for smaller molecules that can be confused with the correct molecule.

    The charged incorrect amino acid enters active site, then into the editing site and get hydrolized out before tRNA can attach to it.
  23. How does an aminoacyl-tRNA synthetase prevent smaller amino acids from being incorporated to tRNA (post-transfer)?
    An aminoacyl-tRNA synthetase has an editing site for smaller molecules that can be confused with the correct molecule.

    If the hydrolytic between AA and tRNA already occurred, the AA still enters the editing site where the link is hydrolyzed by the synthetase.
  24. What are the two classes of synthetases? What are their differences?
    Class I: recognizes the minor groove on the tRNA and attach AA to 2' OH of terminal ribose

    Class II: recognizes minor groove of tRNA and attach AA to 3' OH of terminal ribose

    (possibly for easier differentiation and recognition)
  25. What is the general location of rRNA and r-protein in the ribosome?
    rRNA generally on the interior, interacting with the tRNA

    r-proteins generally on the exterior

    As the organism becomes more complex, the ribosome grows in size. For example, a eukaryotic ribosome has layers of rRNA (40S/60S - L/S), then r-protein, then rRNA with molecular handels that allow for cell regulation.
  26. How does the protein folding differ from the large and small subunit?
    • Small Subunit - domains fold independently (allows for more flexibility in moving down mRNA)
    • Large Subunit - domains are interwoven (provides stability of the active site)
  27. Extra Note: Function of ribosome reflected by conserved primary, secondary, and tertiary structure
  28. What region of the small ribosomal subunit is responsible for mediating tRNA and mRNA interactions?
    The "decoding region" of the 16SrRNA
  29. What region of large ribosome subunit is the catalytic center or peptidyl transferase?
  30. What sites of the large ribosomal unit require complimentary base pairing with the mRNA?
    P and A sites
  31. How do proteins affect translation (broad picture)?
    Translation is facilitated by protein factors that allow translation to move much faster. Translation can proceed without them but at a much slower rate.
  32. What are the four key steps that occur during translation?
    • 1) Initiation
    • 2) Elongation
    • 3) Termination
    • 4) Ribosomal Recycling
  33. What is the name of class I release factors used by eukaryotes and bacteria during termination of translation?
    Class I Release Factors: mimic A site to promote termination

    • Eukaryotes: 
    • eRF1: used to recognize UAA, UAG, UGA 

    • Bacteria:
    • RF1: recognizes UAA and UAG
    • RF2: recognizes UAA and UGA
  34. Hybrid States Model
    A proposed movement of tRNA in which the tRNA is in contact with at least 1 subunit. The tRNA and ribosome move as 1 single unit all together.
  35. What are two ways translation factors work / act?
    • 1) GTPases (catalyze hydrolysis of GTP to do something)
    • 2) bind to ribosome to prevent inapropriate interactions (like IF1 and IF3 preventing tRNA to bind to E and A sites or large ribosome from binding)
  36. If GTP hydrolysis too slow, reactions can be prompted by...
    GAPs: Attach to GTP and promote GTP to turn into GDP

    • GTP -> GAP -> GDP
    • GDP -> GEF -> GTP

    GEFs: Promotes GDP to exchange to GTP
  37. What region of the ribosome do GTPases primarily interact with?
    Protein region
  38. Role of eIF4A
    Dead Box Helicase that unwinds inhibitory mRNA that impedes ribosome
  39. Role of ABCE1
    Promotes ribosomal recycling through ATP Hydrolysis
  40. Role of EFG (eEF2)
    Mimc tRNA A site to recruit tRNA
  41. What are the key step that occur during intiation of translation? (3)
    • 1) initiatuion codon, AUG, in the ORF (Open Reading Frame) is recognized
    • 2) tRNAfmet loaded into the P site of the small subunit and base pair with initiation codon
    • 3) large subunit gets connected.
  42. What makes the initiator tRNA special (the first tRNA that gets loaded)?
    It does not associate with the transporters EFTu/eEF1A because of the additional formyl group stabilizing it.

    It has an AU in the top acceptor stem, and 3 GC's in the bottom acceptor stem that prevent EFTu or eEF1A from accepting it.
  43. How is the initiator loaded in eukaryotes?
    tRNAfmet is bound by the GTPase eIF2, and gets loaded into P site by it
    How does the bacteria locate the AUG start site?
    The Shine Delgarno Sequence (polypurine tract upstream AUG) base pairs with 16SrRNA of small ribosomal unit and guides AUG into P site
    What is the process of binding the initiator tRNA and what proteins are involved?
    Key Proteins: IF1, IF2, IF3

    • IF2: GTPase that guides tRNAfmet to the P site
    • IF1 and IF3: sit in E and A site preventing from tRNA from going there or large ribosomal subunit from attaching
    What must occur for the large ribosomal subunit to attach?
    IF2 GTPase hydrolysis from GTP to GDP causes large ribosomal unit to attach
  47. What is the main difference between bacterial and eukaryotic initiation?
    Bacteria use the Shine Delgarno Sequence recognized by 16SrRNA 

    Eukaryote initiation starts with mRNA cap and recognized by 18SrRNA
  48. What eukaryotic proteins are involved in initiation?
    • eIF1, eIF1A
    • eIF2, eIF2B
    • eIF3
    • eIF4A, eIF4B, eIF4E, eIF4G
    • eIF5, eIF5B
    • PABP
    What helps guide 5' cap to the poly (A) tail to start codon?
    Kozak sequence before AUG that is recognized (A/G)XXAUGG
    How is the closed loop complex formed?
    eIF1, eIF1, eIF2, eIF2B, eIF3, eIF4A, eIF4B, eIF4E, eIF4G, eIF5, eIF5B, PABP

    • eIF4 helps create closed loop structure
    • 1) PABP binds to poly (A) tail and eIF4E bound to mRNA cap
    • 2) eIF4G is the bridging protein of the two
    • 3) eIF4A and eIF4B associate with eIF4E but will be used for scanning later on
    After the close looped complex is formed, how does scanning for start site occur?
    eIF1, eIF1A, eIF2, eIF2B, eIF3, eIF4A, eIF4B, eIF4E, eIF4G, eIF5, eIF5B

    eIF4A and eIF4B scan the mRNA for the AUG start site

    eIF3 and eIF4G interact with small ribosomal unit to bring it to eIF4E
    What happens when eIF4A and eIF4B locate the translation start site, AUG?
    eIF5 functions as a GAP and hydrolyzes eIF2 triggering eIF release and large ribosolmal subunit to join.

    Only the eIF4 family stays on.
    What proteins are involved in loading the tRNA to the correct site in small ribosomal unit?
    eIF1 and eIF1A bind to E and A site

    eIF2 is a GTPase that guides correct tRNA to P site
  54. What are the 3 key steps that occur during the elongation process of translation? Proteins involved?
    Proteins Involved: eEF1A (EFTu), eEF2 (EFG)

    • 1) Decoding: eEF1A (eukaryotes) and EFTu (bacteria) load tRNA to the A site of the ribosome (GTP hydrolysis used to evaluate correct pairing)
    • 2) Peptide Bond Formation: The growing polypeptide chain is transferred to the aminoacyl tRNA in the P site, and the peptide transfer center catalyzes the formation of the peptide bond (driven by ATP during AA activation)
    • 3) Translocation: eEF2 (eukaryotes) and EFG (bacteria) promote the 3 nucleotide base movement along the mRNA so tRNA in A site is in P site (translocation) and the tRNA in P site moves to the E site to exit
    What are the basic steps that occur during the decoding step of elongation?
    • 1) EFTu (GTP) or eEF1A (GTP) chaperone the tRNA with the proper anticodon into the A site
    • 2) small ribosomal element evaluates codon-anticodon complex
    • 3) if the correct confirmation is made, ribosome acts as a GAP causing EFTu / eIF1A to hydrolyze, so nucleotides A1492, A1493, G530 undergo confirmation change, allowing tRNA to fully accomodate A site
    Why does a cognate tRNA increase fidelity during decoding step of elongation?
    Cognate difference in energy is a thermodynamic contributor to fidelity.

    • Proofreading:
    • 1) A cognate associates more tightly to mRNA and is less likely to fall off
    • 2) Cognate GTPase activity much faster, and less likely to fall off
    What are the basic steps that occur during the peptide bond formation step of elongation?
    • Peptide chain is transferred to aminoacyl tRNA
    • -Peptidyl transferase catalyzes peptide bond formation by orienting tRNA for nucleophilic attack
    What are the basic steps that occur during the translocation step of elongation?
    1) acceptor end of tRNA moves to P site

    2) EFG or eEF2 catalyze movement of anticodon end through GTP hydrolyses (both mimic tRNA structure to promote movement)

    Image Upload 4
  59. What are Class I release factors in eukaryotes and bacteria? What do they do?
    • Eukaryotes: 
    • eRF1 (for all 3 stop codons)

    • Bacteria:
    • RF1 - UAA, UAG
    • RF2 - UAA, UGA

    Release Factors recognize a stop codon in the A site and cause a hydrolytic reaction by the peptidyl transferase to release peptide chain (so they are bifunctional)
    What are the class 2 release factors? What do they do?
    RF3: GTPases that promote termination. 

    • 1) Appears after the peptide is released which was promoted by RF1 or RF2
    • 2) GTP hydrolysis of RF3 causes dissociation
    How does termination occur?
    • 1) eRF3 escorts eRF1 to A site
    • 2) eRF3 hydrolyzes and removes itself from eRF1
    • 3) ABCE1 (ATPase) promoted to eRF1 and as it is hydrolyzed, it causes peptide release
  62. What are some potential things that cause a mismatch to occur? How is it fixed?
    • -frameshift miscoding
    • -depleated aminoacyl-tRNA

    • 1) A mismatch causes petribution at the P site which decreases fidelity and promotes premature termination by recruiting RF
    • 2) Once the peptide is terminated, it is degraded by cellular peptidase
    What are the key steps in ribosomal recycling?
    • 1) Class 1 RFs dissociate with the help of RF3
    • 2) RF3 hydrolyzes and dissociates

    • 3) RRF with EFG wedges in between small and large ribosomal subunit
    • 4) GTP hydrolysis of RRF causes dissassembly of subunits
    • 5) IF3 binds to small subunit to stabilize it

    *Sometimes bacteria ORF to close to be released, so ribosome stays on*
    What are the key steps in ribosomal recycling?
    • 1) After the peptide is released, eRF1 stays bound to the A site and promotes ribosomal dissociation
    • 2) eRF1 exchanges with ABCE1 (ATPase) that promotes dissociation through ATP hydrolysis

    3) eIF1A, eIF1eIF3 bind to small ribosome to ensure stability
    What is the bacterial response to low levels of aminoacyl tRNA?
    There are higher level of tRNA (uncharged) that begin to compete for the A site.

    • 1) uncharged tRNA in A site recruits Re1A and causes synthesis of ppGpp using GTP
    • 2) ppGpp decreases the number of tRNAs present and induces stress factors to replenish aminoacyl tRNA
    What is the eukaryotic response to uncharged tRNA?
    • 1) Uncharged tRNA bind to Gcn2, that has a histidyl-tRNA synthetase (HisRS)
    • 2) HisRS phosphorylates eIF2, increasing affinity for GEF eIF2B and depletes eIF2 concentrations
    How doe eIF2B regulate eIF2?
    eIF2B acts as a GEF, and switches GDP on eIFT to GTP, activating eIF2

    If eIF2B was phophorylated, it would remain stuck to eIF2
    What is the effect of eIF4E and how can it be regulated?
    eIF4E is attracted to the mRNA cap for initiation of translation, ESPECIALLY phosphorylated.

    4E-BPs attach to eIF4E and decrease eIF4E affinity for mRNA cap. eIF4E activity restored when 4E-BP phosphorylated.
  69. What are the driving regulatory regions in bacteria and eukaryotes?
    Bacteria: Shine-Delgarno seqeunce

    Eukaryotes: 3' UTR (prevent close loop and cause global repressions)
    How can polyadenylation be regulated by 3' UTR (how is it repressed, and how is repression cured)?
    In dormant mRNAs, they have small poly (A) tails before they are matured.

    • 3' UTR's have cytoplasmic adenylation (CPE's) that affect translation
    • -CPE bound to CPEB, translation become repressed
    • -CPEB is bound to Maskin that interacts with eIF4E preventing it to interact with eIF4G

    • Activation of Dormant mRNA:
    • 1) Phosphorylation of CPEB increases affinity for CPSF
    • 2) CPSF binds to polyadenylation signal and recruits poly (A) polymerase
    • 3) polymerase etends poly (A) tail
    • 4) extension recruits PABP that recruits eIF4G and displaces Maskin from eIF4E
    How does DICE affect transcription?
    In 3' UTR, it binds to hnRNPK and E1/E2 proteins preventing ribosomal units from joining.
    How does Nano affect translation?
    Nano is regulated by 3' UTR TCE. TCE bound to smaug interacts with Cup, and 4E-BP, preventing translation.
    How does miRNA regulate translation?
    miRNA are small RNA's that can bind to the 3' UTR and recruit proteins that inhibit translation.
  74. Defien Chaperones
    Proeins that assisst in proper folding of the protein by prenventing improper folding because the sequence is not enough to induce proper folding.
    What are main bacterial chaperones, and how do they work?
    Trigger Factors that have hydrophobic regions that safeguard protein from misfolding and aggregation.
  76. How does Hsp70 proteins assist in folding?
    They clamp around hydrophobic regions of polypeptide (DnaK). Binding of ATP causes clamp to release.
  77. How does Hsp60 assist in protein folding?
    They form barrel like structures around the polypeptide to prevent misfolding and agregation.
  78. Signal Motiffs
    • Signal sequences within proteins that target proteins to localization machinary.
    • -usually around N or C terminus
    • -can target specific polypeptide chain or a chain with specific characteristics
    • -proteins can have more than 1
    • -typically cleaved
  79. How do Signal Recognition Particles (SRPs) work?
    • SRPs: recognize hydrophobic signal at exit tunnel
    • 1) SRP bind to sequence
    • 2) SRP interact with SRP receptor and hands off polypeptide to Sec A (Sec 61 in eukaryotes) driven by ATP
    • 3) translocase (above) guides to destination
  80. Nuclear Pore Complex (NPC)
    Allow inner and outer transport of macromolecules.
  81. How does RanGTP affect import and export?
    RanGTP creates directionality through the Nuclear Pore Complex (NPC).

    • -Attached to GTP, it allows import protein to drop off cargo
    • -Attached to GDP, it moves export proein out, and hydolyzed it releases cargo
  82. Protein Kinases
    • Transfer ATP phosphate group to protein via phosphorylation. (Protein phophotases do the opposite)
    • -Ser, threo, tyr typically phophorylated in eukaryotes
    • -His, aspartate typically in bacteria
  83. What affect does phosphorylation have on protein? (2)
    • -creates a binding site
    • -changes protein activity
  84. Bacterial 2 component system
    A signal cause intermembran to phosphorylate it self on the inside which the phosphorylates response regulator.
  85. 3 Proteins in Ubiquitination Cascade
    • E1: UB activating
    • E2: UB conjugating
    • E3: UB ligating

    • RING - single jump
    • HECT - move Ub from E2, to E3, to substrate
  86. K-48
    Targets proteins for degredation, straight triangle line of 4.
  87. K-63
    Linked to damage repair and is staggered triangles.

    Recruits BRCA1
  88. What protein causes protein degredation?
  89. Unfolded Proten Response (UPR)
    Cells sense unfolded protein via IRE1, PERK, ATF6 and release BiP that binds to unfolded protein, but also send signal to stop transcription.
Card Set
Molecular Bio Exam Questions
Molecular Biology Final Exam