13: ribosome

Question 1

Aminoacyl-tRNA role

Answer 1

provide amino acid building block for ribosomes

Question 2

tRNA Structure recap

Answer 2

2D=Cloverleaf
3D=b.p stem with non b.p loop regions
> 3’CCA End: Acceptor Stem, AA add here
> recognition sites for tRNA synthesis

Question 3

how many tRNA genes for how many anticodons?
+ how many total anticodons

Answer 3

Around 400 genes for 47 Anticodons
> there are 61 anticodons

Question 4

How are there more anticodons than tRNA anticodons loops?

Answer 4

the same tRNA codon can recognise multiple anticodons due to
> 3rd allowing wobble base pair :>A

Question 5

Aminoacyl-tRNA synthetases (aaRS) 2 steps and what’s special abt it (2)

Answer 5

1. AA activated by phospho adenylation with ATP
2. AA transferred to CCA end of tRNA
   > only loads correct AA = inc fidelity
   > Separate editing and synthesis site (add AA vs Hydrolysis of incorrect bond) (similar to DNA polymerases)
   > increased ATP req tho

Question 6

Aminoacyl-tRNA syunthetases classes

Answer 6

Class 1:
2 sequence motifs
Directly recognise anticodon
Amino-acylation at 2’OH (then migrates to 3’)
Larger and Hydrophobic
e.g. Arg, Cys, Gln, Val

Class 2:
3 sequence motifs
Indirectly recognise anticodon (thru other elements)
Amino-acylation at 3’OH
Smaller and Hydrophillic
e.g Ala, Asp, Gly, His, Ser

Question 7

aaRS class structures?

Answer 7

Large structural variability and diversity within the classes

Evolutionary diverse too :>

Question 8

Ribosome types: 3 IMP and ?

Answer 8

Bacterial, Eukaryotic, Mitochondrial

general core func parts conserved in evo

Question 9

Bacterial Ribosomes:

Answer 9

70S
> 50S L. subunit (2)
> 30S S. subunit (1)
= 3 rRNA

> MW. 2.3MDa
54 Proteins

Question 10

Eukaryotic Ribosomes
vs bac?

Answer 10

80S
> 60S L (3)
> 40S S (1)
= 4 rRNA

More proteins (80) and higher MW (3.3+) than bacteria

Question 11

Mitochondrial ribosomes

Answer 11

different to bacterial
> Translate mem. protein subunits for respiratory chain = Specialised

=2 rRNA

Question 12

Microsomal particles

Answer 12

was what they first called ribosomes until 60s
> TL mRNA into protein

Question 13

Steps in structural studies of ribosomes:

Answer 13

1. Disassociation of 80S ribosome into subunits (60/40) with low Mg conc
2. Negative EM
   = surface, no subunits
3. Immuno-EM
   = surface protein mapping
4. Gel electrophoresis
   = Protein analysis, identification, seq, crosslinking
5. In Vitro reconstruction (w/ the isolated proteins)
6. CryoEM
   = low res, then high after crystallisation by Yonath
7. X-ray crystallography !!
   = from 9 then up to 2.4A

Question 14

Conclusions from 50S subunit X-Ray crystallography

Answer 14

Ribosome = Ribozyme
Ribo proteins = unusual struc. wise
Ribosome rRNA has domains

Question 15

What are Polysomes + key feature for fidelity

Answer 15

assembly of ribosomes for the synthesis of 1 mRNA
> maximised distance between exit sites to prevent misfolding

Question 16

70S ribosome structure and dynamics

Answer 16

2 conf states observed
head region of S. subunit swivels

subunit interface = rRNA surrounded by protein bridges w/ Mg2+ ions

Question 17

Expressosome def

Answer 17

Ribosome + RNA poly + mRNA

Question 18

80S ribosome, vs 70S

Answer 18

resembles 70S = core regions closely related to bacterial
Expansion segments in periphery = different to bac

40S and 60S subunits also structured

Question 19

how were the 40S and 60S subunits structured?

Answer 19

with help from protein factor: elF6
> prevents premature association and inc stability

without it they wouldn’t be structured

Question 20

Eukaryotic vs Bacterial ribosome structure

Answer 20

Has expansion elements in solvent exposed side

however subunit interface is similar and conserved

Question 21

Eukaryotic vs Bacterial ribosome protein interacxtions

Answer 21

Additional proteins in eukaryotic engaged in extended protein-protein interaction networks !!

Bacteria = Isolated and only interact with ribosome RNA

Question 22

Mitochondrial ribosome characteristics (Yeast and mammlian)
function (2)
evo (2)
structure (2)

Answer 22

Specialised in synthesis of membrane proteins for respiratory chain
= Adapted regulation and reduced genetic code

Evolution:
Larger nu. of proteins , divergent evo from bacterial !
Lack 5S rRNA (only 2 rRNA unlike 3 in bac)

Structure determination:
ONLY with cryoEM
> cant be crystallised for x-ray crystallography so took its time

Question 23

Mito-ribosomes structure vs Bacteria

Answer 23

Conserved t and mRNA binding sites

Remodelling of CP (Central protuberance)
> has mito-specific protein subunits, not present in bacterial

Question 24

Role of S. subunit

Answer 24

Binds mRNA and tRNA , and here is where decoding happensR

Question 25

Role of L. subunit

Answer 25

Host peptidyl-transferase centre PTC and peptide exit tunnel

Question 26

Bacterial Ribosome Cycle 4

Answer 26

1. Initiation 2. Elongation with EPA (e= where tRNA enter, p=pre PTC, a=post PTC) 3. Release = after a stop codon enters A, tRNA then leaves E 4. Recycling

Question 27

Decoding mechanism in bacterial ribosomes where, feature and result?

Answer 27

In S subunit Involves a coupled conformational change of the ribosome RNA upon A-site tRNA binding = Monitors correct base pair and inc fidelity

Question 28

Decoding ribo RNA structure

Answer 28

Tightly monitored watson-crick bp at position 1 and 2 Position 3 = less strict = wobble b.p > 1 tRNA can read many codons with same 1/2 seq but diff 3rd

Question 29

Decoding Local conformation change?

Answer 29

results into global structural transitions

Question 30

Peptidyl transferase Catalysis where how fidelity rate inc with ribosome?

Answer 30

on L subunit No protein in vicinity of CCA end of tRNA in PTC = Inc fidelity Inc rate by 10^5 with presence of ribosome = tRNA itself is the catalysis = does it by optimising allignment/arrangment

Question 31

Peptidyl transferase reaction

Answer 31

1. alpha-amino of tRNA (adding) attacks ester bond in peptidyl t-RNA in P site 2. Proton shuffle aided by H-bond network there is tet. IM and Oxyanion n yea

Question 32

How did we know the PTC reaction

Answer 32

By experimentally resolving structures in their individual states

Question 33

where are the nascent protein and deacylated tRNA after PTC?

Answer 33

Nascent protein = A site of tRNA Deacylated tRNA = P site EPA there are P/E and A/P hybrid states

Question 34

Translocation in Bacterial ribosomes

Answer 34

tRNA must move by 1 codon 1. tRNA movement relative to 50S 2. GTP Hydrolysis in EF-G 3. EF-G cat. movement of 30S has to happen before addition of next tRNA

Question 35

What happens at the exit tunnel in vitro vs reality + e.g + how does protein emerge from exit tunnel

Answer 35

Protein emerges vectorially from exit tunnel In vitro: Unfolded protein wants to rapidly fold into compact states In vivo/reality: Chaperones in place to prevent premature folding e.g. Trigger factor

Question 36

Protein folding at exit tunnel

Answer 36

Ribosome synth. 10 AA/s > Incomplete proteins do not fold tho due to chaperones > they are prone to aggregating tho

Question 37

What prevents protein folding at exit tunnel

Answer 37

Ribosome tunnel + Chaperone trigger factor = Protected env for nascent protein

Question 38

Exit tunnel shape

Answer 38

It prevents premature folding > only after 30 AA added, it permits helical segments at its end

Question 39

Trigger factor

Answer 39

Forms a cradle at the exit for nascent proteins = slows down folding = permits allowed interactions (e.g w/ processing factors) > nothing too strong to induce folding tho ya

Question 40

Antibodies and targeting ribosomes?

Answer 40

Good to target as ribosomes are vital for ALL organisms and there are similarities but also differences = preferred target

Question 41

How can it target bacterial ribosomes? Medi examples breh

Answer 41

There are 3 ways antibodies could target the ribosome 1. By blocking the exit tunnel of the 50S Subunit/L > Macrolides, Strep B 2. By binding to the decoding centre of the 30S > Aminoglycosides

Question 42

Why is it imp to understand the mode of interaction with antibiotics and ribosome targetting?

Answer 42

it helps to make sure it's selective for bacteria and doesn't cause UAW by affecting eukaryotic ribosomes !