文档介绍：Directed hydroxyl-radical cleavage
Datwyler & Meares, TIBS 25, 408 & 468
(S)-1-(p-bromoacetamidobenzyl) EDTA (FeBABE)
Ascorbate
H2O2
Positioning of IF3 relative to tRNAi and IF1 (Dallas & Noller, Mol. Cell (2001)
Model of tRNAMetf relative to IF3, IF1 in A-site and mRNA in P-site
Model for the interaction of the anticodon of fMet-tRNA with mRNA
Notice change in shape of IF3
mRNA decoding
Structure of IF2 (archaeal homolog of eIF5B)
Roll-Mecack et al., Cell 103, 708 (2000)
IF2 (eIF5B) binds to the small ribosomal subunit
Approximate positioning on ribosome from footprinting data
Domain II of bacterial IF2 interacts with IF1
Promotes binding of fMet-tRNAfMet to small ribosomal subunit
GTP hydrolysis upon release from ribosome and subunit joining
Summary Prokaryotic Initiation
There are plenty of functional and structural data
Ribosome - structure and active site
IF1 location - place holder -directs fMet-tRNAi to P-site
IF3: aids ribosome dissociation location adjacent to tRNAi
IF2 location mapped - needed for 50S joining
Order of assembly still unclear
Mechanism of 50S joining ?
Eukaryotic Initiation - big picture
Recruit Met-tRNA to small particle
Recruit mRNA to small particle
Scan to AUG codon
Bind large particle and release initiation factors
Elongation starts
1.
2.
3.
4.
5.
Three connected processes
Build up of small ribosomal particle
Build up and delivery of plex (eIF2/fMet-tRNAi/GTP)
Build up and joining mRNA/initiation plex
Eukaryotic Translation Initiation - details
Dissociation of ribosome binding eIF1A and eIF3 to 40S
Recruit plex (fMet-tRNAi, eIF2, GTP) to 40S
Recruit mRNA to 43S -> 46S particle
46 S scans mRNA to reach AUG
Initiation factors leave and 80S joins
Elongation begins
Highly controlled process
Loss of control -> cancer
Eukaryotic Translation Initiation - Details
Dissociation of ribosome binding of
eIF1A (A-site) and eIF3 to 40S
eIF3 is a plex of 11 subunits (MW = MDa) - not homologous to I