Shine Dalgarno sequence

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Shine Dalgarno sequence
Purine rich

• Start of protein synthesis
• 3-end of 16S rRNA binds Shine Dalgarno sequence
• Initiation codon binds anticodon of initiation
  tRNA.

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Start of protein chain

• First residue in protein chain (of prokaryotes)
  is a formyl-methionine
• (while other methionines are not formylated).
• Initiation tRNA
• Two different Met-tRNA tRNAf and tRNAm
• One Met-tRNA synthetase
• Enzyme that formylates exclusively
• Met-tRNAf
• Why is a special fMet used?

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(No Transcript)
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Formation of initiation complex

• Steps in initiation
• 30S subunit with IF1 and IF3 bound
• (IF1 and IF3 prevent premature binding of 50S
  forming a dead-end complex)
• 30S binds Shine-Dalgarno sequence of mRNA
• fMet-tRNAf binds initiation AUG
• (delivered by IF2)
• IF1 and IF3 leave
• 50S binds forming the 70S initiation complex
• (which determines the reading frame)

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Elongation is a 3-step process
Out protein chain
In mRNA-chain
3 binding sites for tRNA on the ribosome A
aminoacyl P peptidyl E exit
In the P en A sites the tRNAs bind to mRNA and
both ribosomal subunits
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Protein leaves 50S subunit through a tunnel
Tunnel allows passage of unfolded or possibly
helical protein chains. Folding of the protein
happens after exit of the tunnel (with help of
chaperones).
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Elongation process (1)
Formation of peptide bond in peptide-transferase
center (formed by rRNA)
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Peptide bondformation
Excellent comprehensive review of ribosome
functioning !!
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Elongation process (2)
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Selecting tRNAs
Chemical modification of Cys in tRNACys to Ala
shows that only the anti-codon determines which
amino acid is incorporated.
This aspect is used to incorporate non-natural
amino acids ! (however, only L-amino acids can be
incorporated)
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Codon-anticodon recognition
tRNA
mRNA
Testing of correct pair on minor groove
side (check present only on 1rst and 2nd
positions)
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wobble

• XYU and XYC mostly code
• for the same amino acid
• - XYA and XYG idem dito.

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Exact codon-anticodon pairing would require more
tRNAs Example it would require 6 tRNAs for Leu
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Elongation delivery of tRNA requires EF-Tu

• EF-Tu brings aminoacyl-tRNA to mRNA and protects
  labile ester bond between tRNA and a.a. from
  hydrolysis
• EF-Tu checks correct docking, correct interaction
  of codonanti-codon. Correct docking results in
  GTP to GDP P hydrolysis and release of EF-Tu
• GTP loading of EF-Tu performed by EF-Ts
• EF-Tu doesnt binds initiation fMet-tRNAf.
• Vice versa, IF2 does not recognize Met-tRNAm.

43 kDa G-protein
Elongatie factor Tu
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Elongation translocation requires EF-G
Elongation factor G (translocase) structurally
mimics the EF-Tu tRNA complex.
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Termination requires release factors

• Elongation continues untill a stop codon (UAA,
  UGA or UAG) is encountered on the mRNA
• No tRNA recognizes stop codons
• Stop codons are recognized by specific proteins
  release factors RF1, 2 and 3

Important function of the ribosome is to protect
the fragile ester bond between the growing chain
and the tRNA. Chain termination is achieved by
allowing hydrolysis by water.
Dissociation of 30S, 50S, RNA en mRNA by
GTP-dependent RRF (ribosome release factor)
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Release factor induces hydrolysis, which results
in formation of a COO- terminus
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Eukaryotic protein synthesis
Scanning for AUG (helicase)

• Compared to prokaryotes
• Ribosomes are more complex
• (60S 40S 80S ribosome)
• Initiator tRNA is met instead of fmet
• with specific initiator tRNA (met-tRNAi).
• Initiation codon is always AUG geen
• No purine-rich Shine-Dalgarno sequence,
• Initiation always starts on first 5 AUG
• found by walking (ATP dependent) over
• the mRNA from the 5 end side

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Eukaryotic protein synthesis

• Circularized arrangement
• 5 cap and poly-A tail
• eIF-4E, eIF-4G and PABPI
• Large number of initiation factors
• EF1? EF-Tu (EF1?.GTP brings tRNA to
  ribosome
• EF1?? EF-Ts GTP-GDP exchange factor
• EF2 EF-G translocatie factor
• eRF1 is the (single) release factor
• eIF3 IF3 prevents reassociation of subunits

mRNA codes for only 1 protein (not
polycistronic) thus only 1 initiation site
(first AUG) per mRNA.
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Antibiotics
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Protein targeting
Signal sequence directs ribosome to cytoplasmic
side of the Endoplasmatic Reticulum (ER)
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• Cytoplasm
• - Signal sequence recognized by
  signal-recognition particle (SRP
• Docking on to SRP receptor
• Translocon transmits protein chain across
  membrane
• ER lumen
• Signal peptidase
• Folding chaperones
• Glycosylation
• Budding for transport via vesicles

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Cargo receptors direct proteins Budding by
coat proteins (COPs and others) Membrane dusion
by SNARE proteins v-SNARE (vesicle) and T-SNARE
(target)