Lecture 9- Translation and targeting Flashcards
relationship between ribosomes and the nuclear pore
can accommodate both subunits in all orientations for transport- just about
why is targeting so important
ensuring proteins can get to the right organelles, parts of cells etc
what is the current model of co-translational insertion
ribosomes directly moving peptides into the ER, where they can then be appropriately folded- this allows proteins that would be too big for the pores to get in
pulse chase analysis- principle and application
radioactive AAs used to label cells
more radioactive AAs added to ‘chase’, following this provided evidence of secretory proteins being transported
reconstitution experiments
addition of labelled puromycin- which contributes to protein synthesis- this can then be seen in the ER
what is the signal hypothesis
idea that signalling, such as on the membrane of the ER, is important in where ribosomes are allocated
milstein and brownlee
found that the correct assembly of IgG light chains required signals from part of the ER, could also identify signal sequences from immature precursor polypeptides
another way of proving the importance of s signal sequences
adding signals to molecules which are not usually involved, can make excretion happen
how are transmembrane domains structured
opposite polar regions at each end, with 20ish hydrophobic amino acids in the middle
SRP
signal recognition particle, which allows translocation across/insertion into the ER membrane
how SRPs contribute to translocation
SRP binds to ribosome, pausing transcription
SRP binds to SRP receptor on the membrane
ribosome can then bind to the protein translocator
SRP displaced
components of the SRP
RNA/peptide units
SRP54- signal recognition
SRP9, 14- translational arrest
SRP68, 72- ER docking
what other mechanisms can SRPs use
negative selection, e.g. binding to proteins that are not going to the ER
how does the SRP stop binding
SRps 9 and 14- conformational changes causes SRP to compete with the elongation factor at the ribosome
how is NAC involved in the process of SP recognition
NAC (nascent polypeptide associated complex) associates with emerging polypeptides, but associates more with hydrophilic sequences, leaving hydrophobic sequences more free for SRP recognition
how do polypeptides get across the membrane- mechanism
ribosome membrane fusing with the ER membrane
chain terminates and is released- puromycin causes this, and has been shown to be necessary
mutant used to identify the Sec61 translocon
His4 mutant yeast, which cannot make histidine
can add back ER targeted His, look for mutants where they can still grow, and genes can be identified- e.g. Sec61
how else has Sec61 been identified
found that addition of Sec proteins could make an artificial liposome functional
how is Sec61 structured and how does this aid its function
‘plug’ which becomes displaced, interchange for signal sequence binding with negatively charged residues, signal sequence binds here, rest of thr polypeptide can be moved in
translocation system in prokaryotes
SecYEG- same mechanism, but through the plasma membrane
example of how insertion can be mediated in eukaryotes
BiP proteins- can bind to peptides and ‘pull’ the proteins through into the membrane from the inside
example of how insertion can be mediated in prokaryotes
SecA ATPase, can drive translocation in bacteria by forming more of a ‘clam’ structure on the ourside, rather than the inside, of the membrane
