Lecture 21

Question 1

Fragment reaction mimics peptidyl transfer

Answer 1

get peptide chain reaction in test tube

Question 2

fragment reaction reveals

Answer 2

peptidyl transferase enzyme is RNA not protein
80% activity- worked almost normally with just RNA
used rnase, totally killed activity
suggests that RNA part is doing the catalysis
he was conservative; but we know he was right now

Question 3

structure confirms noller result: ribosome is a ribozyme

Answer 3

no proteins within 18 ang of peptidyl transfer active site: PT center within the largest ribosomal unit
no protein near- just rna, more to support his conclusion

Question 4

Elongation: many antibiotics bind in peptidyl transfer center (PTC)

Answer 4

toxins that stop protein synth attack ribosome- can be targeted for good and bad
tetra cyclone: binds 30s A site; prevents aa-tRNA entry
puromycin: binds in ptc, chain terminator
chloramphenicol: binds in PTC, inhibits peptidyl transfer reaction and blocks exit tunnel
macrolides: bind in PTC, block exit tunnel

Question 5

Elongation cycle: the hybrid state model of elongation

Answer 5

selection/accomodation
peptidyl transfer
translocation
gtp hydrolysis at selection/accomodation (EF Tu)
also at translocation (EFG)

Question 6

Elongation Cycle: translocation is powered by EF-G, a GTP powered motor

Answer 6

Ef Tu: GTP + aa-tRNA is for decoding
EF-G: GTP is for translocation
look really similar
all RNA on the left, all protein on the right
elongation factors have evolved to look like tRNAs
makes sense b/c they need to bind to ribosome
can displace EF Tu which is only loosely bound at this point

Question 7

Elongation cycle: the hybrid state model of elongation

Answer 7

Ef Tu and egg look almost identical; why this works

both need to be able to bind to the a site

Question 8

Review of elongation

Answer 8

a site: tRNA selection
aa-tRNA: EF-Tu: GTP

P site: peptidyl transfer
ribosome large subunit

translocation: Uncharged tRNA exits from E site
EF-G: GTP

Question 9

Termination: release of polypeptide from tRNA in P site

Answer 9

Finish with stop codon; codon for which there is no tRNA that recognizes it
release factors: things that recognize stop codons
don’t need to know names
they can bind; don’t have aa, instead of having nuc attack, now will just have water acting as a nucleophile
full ribosome assembled. will separate large and small subunits. extra steps to term

Question 10

Termination: EFG cooperates with release factors to disassemble spent tRNAs, subunits, mRNA

Answer 10

extra steps involve more action from EFG
uses energy
ribosome releasing factor hydrolyzes gtp as a signal to disassemble everything. breaks apart, back to where you started

Question 11

Termination: release factors shaped like tRNA

Answer 11

ribosome releasing factor also shaped sort of same way as decoding and translocation

Question 12

Ribosomes and protein synthesis

Answer 12

know that release factors disassemble complex and that they use a little energy to do that
occupying the a site; prevent complex from forming until ready

Question 13

moving on: what happens to proteins after they are made

Answer 13

the n terminus of the growing polypeptide worms through na exit tunnel in the large subunit
protein folding has to wait until out of exit tunnel- big enough to hold 30aa
soluble protein will get released and finish it’s folding–???

Question 14

Examples of protein targeting signals

Answer 14

proteins have signal that indicate where they should end up
sig seq usually just refers to signal to go to ER and go through signal pathway
nucleus: tends to be internal b/c maybe want to hide a certain signal

Question 15

To target ER

Answer 15

N terminal or internal

signal sometimes removed

Question 16

Mitochondrial matrix

Answer 16

N terminal (multiple)
signal removed

Question 17

Peroxisome

Answer 17

C terminal

signal not removed

Question 18

Nucleus

Answer 18

Internal
signal not removed
b/c a lot of those proteins go back and forth
also nuc envelope gets broken down during mitosis; if there were no seq then they wouldn’t be able to get in

Question 19

Nuclear localization signals direct proteins to the nucleus

Answer 19

NLS is located internally and is not cleaved off

Question 20

Proteins are targeted to different compartments in different ways

Answer 20

enters sec pathway: ER, then can go to golgi, golgi sends out to lots of diff places
secretion: here to plasma membrane, where all secreted proteins go

Question 21

But what about membrane or secreted proteins?

Answer 21

Membrane targeting: cotranslational protein translocation
synth on ribosomes attached to ER
rough ER
exit tunnel of ribosome docked into complex
threaded through, straight into the middle of the lumen
question: will this drag entire ribosome over to ER? yes

Question 22

Pre protein translocate (Sec61 complex) a protein conducting channel with an aqueous pore that spans the ER membrane

Answer 22

Pre protein translocate is an integral membrane protein. it therefore requires other molecules of pre protein translocase for its correct insertion into the ER membrane
Plug is on the lumen side. When ribosome docks, plug is removed.

Question 23

Membrane targeting: ribosome docked to pre protein translocase

Answer 23

tRNA binds, nascent chain binds, Sec 61 bound

Question 24

Membrane targeting: N terminal hydrophobic signal sequences direct secreted and membrane proteins to the translocase

Answer 24

Long run of hydrophobic things: structurally, that has features that can be recognized as signal seq to enter sec pathway
often cleaved off, does not form a mature protein
positive charge, followed by hydrophobic stretch of about a dozen residues, followed by cleavage site
sig seq are molecular zip codes

Question 25

Membrane targeting: signal recognition Particle

Answer 25

SRP has three functions:
recognize signal sequence emerging from ribosome exit tunnel
Pause translation
-stop translation; trying to shove into ER, not cytoplasm
-drag entire ribosome with chain over to channel
-on translocon, resumes synthesis
Direct ribosome and nascent polypeptide to pre protein translocate channel on ER membrane
Like the ribosome and the spliceosome, SRP is a ribonucleoprotein

Question 26

Membrane targeting: SRP reaches from the exit tunnel to the A site

Answer 26

Membrane targeting: SRP cycle
1) SRP receptor binds to pre protein trnaslocase Sec 61 complex
2) SRP binds signal- translation paused
3) SRP receptor and SRP come together. neither gtp binding protein can hydrolyze gtp on its own. once you bring two halves together, then can hydrolyze gtp
5: SRP falls off, translation resumes
gtp binding protein, recognize structural features (hydrophobic sig seq)
sitting on translocate: SRP receptor
also gtp binding protein
Pushing chain through translocon

Question 27

Now we see why the region around the ribosome exit tunnel is highly conserved

Answer 27

its got to interact with SRP and the pre protein translocate

makes sense b/c this part of the ribosome has to not just interact with SRP but also sit on translocase
doing a lot of jobs

Question 28

Membrane targeting: signal peptidase cleaves the signal peptide

Answer 28

Sig seq can hold onto hydrophobic part and thread the rest of the protein through
once through, signal peptidase cleaves of signal sequence
release protein inside ER
nuclear signal seq- why would you not wanna throw away?
during cell div, dissolve the nucleus. if your proteins didn’t have this it would be a problem
also some proteins going to be shuttled back and forth

Question 29

membrane targeting: integral membrane proteins

Answer 29

start vs stop transfer
cuts signal peptide off
ends up with single pass transmembrane protein
can flip whole topology of protein around
how does it know how to do this? can only hold onto a couple of these

Question 30

membrane targeting: pre protein translocase allows lateral escape of signal peptides and transmembrane segments into the membrane

Answer 30

okay

Question 31

the secretory pathway

Answer 31

40-50% all proteins will enter the sec pathway
hormone receptors, etc.
ion channels and transporters
30% of the genes in your genome encode membrane proteins

Question 32

The secretory pathway also inserts and folds integral membrane proteins

Answer 32

ion channels
nutrient transporters
hormone receptors
NT receptors
cell adhesion molecules