Lecture 9- Translation and targeting

Question 1

relationship between ribosomes and the nuclear pore

Answer 1

can accommodate both subunits in all orientations for transport- just about

Question 2

why is targeting so important

Answer 2

ensuring proteins can get to the right organelles, parts of cells etc

Question 3

what is the current model of co-translational insertion

Answer 3

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

Question 4

pulse chase analysis- principle and application

Answer 4

radioactive AAs used to label cells
more radioactive AAs added to ‘chase’, following this provided evidence of secretory proteins being transported

Question 5

reconstitution experiments

Answer 5

addition of labelled puromycin- which contributes to protein synthesis- this can then be seen in the ER

Question 6

what is the signal hypothesis

Answer 6

idea that signalling, such as on the membrane of the ER, is important in where ribosomes are allocated

Question 7

milstein and brownlee

Answer 7

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

Question 8

another way of proving the importance of s signal sequences

Answer 8

adding signals to molecules which are not usually involved, can make excretion happen

Question 9

how are transmembrane domains structured

Answer 9

opposite polar regions at each end, with 20ish hydrophobic amino acids in the middle

Question 10

SRP

Answer 10

signal recognition particle, which allows translocation across/insertion into the ER membrane

Question 11

how SRPs contribute to translocation

Answer 11

SRP binds to ribosome, pausing transcription
SRP binds to SRP receptor on the membrane
ribosome can then bind to the protein translocator
SRP displaced

Question 12

components of the SRP

Answer 12

RNA/peptide units
SRP54- signal recognition
SRP9, 14- translational arrest
SRP68, 72- ER docking

Question 13

what other mechanisms can SRPs use

Answer 13

negative selection, e.g. binding to proteins that are not going to the ER

Question 14

how does the SRP stop binding

Answer 14

SRps 9 and 14- conformational changes causes SRP to compete with the elongation factor at the ribosome

Question 15

how is NAC involved in the process of SP recognition

Answer 15

NAC (nascent polypeptide associated complex) associates with emerging polypeptides, but associates more with hydrophilic sequences, leaving hydrophobic sequences more free for SRP recognition

Question 16

how do polypeptides get across the membrane- mechanism

Answer 16

ribosome membrane fusing with the ER membrane
chain terminates and is released- puromycin causes this, and has been shown to be necessary

Question 17

mutant used to identify the Sec61 translocon

Answer 17

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

Question 18

how else has Sec61 been identified

Answer 18

found that addition of Sec proteins could make an artificial liposome functional

Question 19

how is Sec61 structured and how does this aid its function

Answer 19

‘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

Question 20

translocation system in prokaryotes

Answer 20

SecYEG- same mechanism, but through the plasma membrane

Question 21

example of how insertion can be mediated in eukaryotes

Answer 21

BiP proteins- can bind to peptides and ‘pull’ the proteins through into the membrane from the inside

Question 22

example of how insertion can be mediated in prokaryotes

Answer 22

SecA ATPase, can drive translocation in bacteria by forming more of a ‘clam’ structure on the ourside, rather than the inside, of the membrane