Williams BCH 444 2014 Lectures 3-6

E co translational signal cleavage is an event

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Unformatted text preview: - signal cleavage occurs before polypeptide translation is completed, i.e., co-translational - signal cleavage is an event associated with rough microsomal membranes - signal is at N-terminus Are free polyribosomes in the cytosol any different from ER membranebound polyribosomes? 9 Characteristics of cleavable signal sequences Note: Proteins with signal sequence attached are designated by the prefix “pre”, e.g., preprolactin, preproinsulin Gunnar von Heijne (1985): -  n-region: highly variable composition and length but usually carries a net positive charge -  h-region: hydrophobic region typically 10 +/- 3 residues -  c-region: generally not charged but more polar than h-region -  residues at -1 and -3 relative to cleavage site must be small and neutral -3 n-region h-region -1 c-region 10 Watson, M.E.E. (1984) Nucleic Acids Res. 12:5145 cleavage site After the signal.......what s next?? 1. Proteins destined for the ER are synthesized (usually) with an N-terminal signal sequence 2. Signal is recognized by Signal Recognition Particle (SRP) 3. SRP binds to SRP receptor on ER membrane - (brings translating polyribosome to the ER). SRP receptor helps target the ribosome to the translocon pore. 4.  SRP is released, ribosome binds to translocon, nascent chain enters translocon. 5. Nascent polypeptide is translocated into the ER lumen and signal is removed by signal peptidase 1 2 4 3 5 11 Another tour de force by the Blobel lab - SRP discovered by Peter Walter Assay: Translate preprolactin mRNA in an in vitro translation initiation system (from wheat germ) in the presence of high-salt-washed rough microsomes (removes bound ribosomes) (observe: no translocation into ER lumen, no signal cleavage) - Purify activity from the salt wash that restores translocation and signal cleavage eluate ex2 eluate ex1 flow-thru salt extract rough mics hydrophobic chromatography Peter Walter 125I-SRP 72 kDa binds to free ribosomes and to polysomes actively translating signal-containing nascent chains 68 kDa Kd binding to ribs = ~5 x 10-5 Kd binding to signal-containing nascent chains = ~8 x 10-9 54 kDa SDS-PAGE SRP (signal recognition particle) (-signal) 19 kDa 14 kDa 9 kDa SRP blocks in vitro translation of nascent chains with signal sequence (no microsomes present) (+signal) translocation activity 12 Walter, Ibrahimi & Blobel (1981) J. Cell Biol. 91:545 Structure and functions of SRP Alu domain S domain 7S RNA in red + yellow Halic, M. et al. (2004) Nature 427:808 Overall functions of SRP 1. Binds to signal sequences and transiently arrests translation - why? 2. Binds to SRP receptor on ER membrane and thereby directs the ribosome-nascent chain complex to the ER translocon channel Subunit functions SRP54 - binds signal sequences, binds ribosome and binds to SRP receptor SRP68/72 - seems to play a structural role as a brace for the hinge 1 region SRP19 - important for SRP assembly SRP9/14 - ribosome binding/elongation arrest 7S RNA - ribosome binding/elongation arrest (both RNA-RNA and RNA-protein interactions), signal sequence binding? 13 How is SRP able to...
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This note was uploaded on 03/27/2014 for the course BCH 444 taught by Professor Mccallan during the Spring '14 term at University of Toronto.

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