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Molecular Biology BIO-5003B > Lecture 13: Prokaryotic Protein Synthesis > Flashcards

Lecture 13: Prokaryotic Protein Synthesis Flashcards

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1
Q

What are the five stages of proteins synthesis?

A
  1. Activation of Amino Acids
    • tRNA aminoacylation
  2. Initiation of translation
    • mRNA and aminoacylated tRNA bind to
      ribosome
  3. Elongation
    • Cycles of aminoacyl-tRNA binding and
      peptide bond formation until stop codon
  4. Termination and ribosome recycling
    • mRNA and protein dissociate, ribosome
      recycled
  5. Folding and post-translational
    processing
    -catalysed by variety of enzymes
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2
Q

What are the requirements of prokaryotic initiation

A
  1. Requires
    a. 30s ribosomal subunit
    b. mRNA
    c. fMet-tRNA (START) (f = formyl)
    d. Initation factors: IF-1, 2 and 3
    e. GTP
    f. 50s ribsomal subunit
    g. Mg2+
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3
Q

What are the stages of initiation?

A
  1. 30s binds to IF-1, IF-3, and mRNA
    a. IF-3 keeps 30s and 50s subunits apart
  2. START codon (5’ AUG) guided to right
    position by “SHINE-DALGARNO
    SEQUENCE” - region of mRNA
    complementary to sequence in 16s
    ribosomal RNA
  3. fMet-tRNA binds to P (peptidyl) site
    alongside AUG
    a. fMet-tRNA is recruited by IF2-GTP
    which binds to 30s subunit, base
    pairing with start codon
  4. Large 50s subunit combines with 30s
    subunit forming “initiation complex”
    a. IF2 hydrolyses GTP
    b. Followed by dissociation of IF-1, 2, & 3
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4
Q

What is the Singe-Dalgarno sequence?

A

Sequence at the 5’ end of mRNA (prior to AUG) responsible for base pairing with sequence on 16s rRNA at the 3’ end, aligning the AUG sequence

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5
Q

What are the 3 sites in a ribosome?

A
  1. Exit site = “E”
  2. Peptidyl site = “P”
  3. Aminoacyl site = “A”
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6
Q

How does elongation take place?

A
  1. Next tRNA binds to A site “via binding of
    EF-Tu” which is bound to GTP
    a. EF = elongation factor
    b. This protects the aminoacyl-tRNA from
    hydrolysis
  2. Tu is first bound to GTP (Tu-GTP)
  3. Once bound to the A site, GTP is
    hydrolysed, which is recycled by EF-Ts
  4. GTP rebinds, forming a cycle
  5. If the codon / anti-codon interaction is
    weak, then before the GTP can
    hydrolyse, the aminoacyl-tRNA can
    dissociate
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7
Q

What are the two purposes of GTP in elongation?

A
  1. Specificity; due to time taken for
    hydrolysis
  2. Protecting aminoacyl-tRNA from
    hydrolysis
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8
Q

How is the first peptide bond formed in elongation?

A
  1. peptidyl transferase activity binds the
    two amino acids together
    a. Catalysed by “23s rRNA (part of
    50s)
  2. First amino acid = P site, second = A
  3. “uncharged tRNA” (deacetylated) the
    exits via the E site
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9
Q

What is the final step of elongation?

A
  1. Moving along the mRNA strand to read
    subsequent codons
  2. Carried out by hydrolysis of GTP bound
    to EF-G (translocase)
    a. hydrolysis moves deacetylated tRNA to
    E site
    b. charged tRNA then moves to P site
    exposing the A site for next charged
    tRNA
  3. Rapid. 2-15 AA’s per second (20 sec for a
    300 AA protein)
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10
Q

What is the hybrid model?

A

When the charged-tRNA passes its AA to the AA in the A site, the large subunit of te tRNA (top end) ‘leans’ into the E site whilst the anticodon region remains bound in the P site. The same is true the charge tRNA in the A site, leaning into the P site.

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11
Q

How does termination take place?

A 
1. Occurs in response to termination 
   codon in the A site
2. First: RF-1 or RF-2 (depending on 
    which termination codon is present) 
    binds to A site (RF = release factor)
3. LEADS TO hydrolysis of ester linkage 
    between nascent polypeptide and 
    tRNA in the P-site = releasing 
    completed polypeptide
4. Finally; mRNA, deacetylated tRNA, 
    and release factor leave ribosome
5. 50s and 30s subunits dissociate - 
    aided by:
   a. "Ribosome Recycling Factor (RRF)"
   b. IF-3
   c. and energy provided by EF-G- 
       mediated GTP hydrolysis
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12
Q

What do release factors (RF) 1 and 2 bind to?

A 
RF-1 = UAA & UAG
RF-2 = UAA & UGA

EUKARYOTES ONLY HAVE ONE RELEASE FACTOR

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13
Q

How is transcription and translation coupled in bacteria?

A
  1. Can be rapid due to clusters of
    ribosomes called “polysome’s”
    a. this allows mRNA to be translated by
    many ribosomes simultaneously
  2. Translation can begin before
    transcription ends
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14
Q

What is the fundamental difference in initiation between prokaryotes and eukaryotes?

A
  1. Prokaryotes: small ribosomal subunit
    recognises shine-Dalgarno sequence of
    mRNA - this could be internal, as in,
    within the body of the mRNA
  2. Eukaryotes: small ribosomal subunit
    recognises 5’ cap structure of mRNA and
    translation initiates from closest AUG
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15
Q

How does initiation take place in eukaryotes?

A 
1. Absolute requirement for cap on 5' 
   end
2. 95% of euk. mRNA, translation begins 
    at 5'-proximal AUG
3. small ribosomal subunit can scan in 
    one dimension on RNA
4. mRNA secondary structures located 
    in 5' untranslated region inhibit 
    translation initiation
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16
Q

What is the “Kozak consensus sequence”Wh

A
  1. 5’- GCC(A/G)CCAUGG -3’
  2. Identified by Marilyn Kozak around
    AUG start codon
  3. Enhances initiation frequency
17
Q

Why is there many IF’s (initiation factors) in mammals compared to prokaryotes?

A
  1. Due to the need to scan separately
    from the need to assemble the
    ribosomal complex
18
Q

What are the two events in eukaryotic initiation?

A
  1. Assembly of transcription apparatus:
    a. “43s pre-initiation complex”
    b. small ribosomal subunit kept apart
    from large one by eIF3
    c. eIF2-GTP binds to charged tRNA
    d. First tRNA = tRNAi met
2. Combining of mRNA to the 43s pre- 
     initiation complex
   a. 5' cap binds to eIF4G and eIF4A
   b. these deal with any secondary 
       structures in the mRNA
   c. mRNA moves along until first AUG is 
       found, initiating translation
19
Q

What are “upstream open reading frames”

A
  1. AUG (start codons) upstream of the
    initial start codon
  2. allows regulation for the start point of
    translation
20
Q

What are internal ribosome entry sites?

A
  1. when the eukaryotic system ‘doesnt have
    to’ scan
  2. e.g., viral RNA doesnt have a 5’ cap
    structure, yet is still translated, so uses
    RES’s, allowing binding of pre-initiation
    complex
  3. It is possible for cellular RNAs to use
    RES’s. E.g., Apoptosis, where 5’ cap
    structure may be removed
21
Q

What is circularization of eukaryotic mRNA?

A

Poly-A tail at 3’ tail binds to 5’ cap structure (E, G, and A) using poly-A-binding protein

Molecular Biology BIO-5003B (30 decks)

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