Proteins and Such for Exam 4

Question 1

tRNAs

Answer 1

clover leaf 2D structure, L-shaped 3D structure
diff tRNAs have diff sequences but same structure
small ~75-95 bases
3 loops (D-loop, anti-codon loop, T-loop) + acceptor stem (ACC) where aa gets attached (and a variable loop)

Question 2

tRNA synthetases

Answer 2

attach correct aa to the appropriate tRNA
HAS to be accurate
“charge” tRNA
2 classes which have very diff structures and ways they bind their tRNAs (recognize different faces of the tRNA)
some edit

Question 3

tRNA synthetase “editing”

Answer 3

1. size exclusion (not rlly editing): large non-cognate aa are exclude from the aminoacylation site
2. editing pre-transfer: smaller non-cognate aa are occasionally activated (to form aminoacyl-adenylate) but are recognized by the editing site and are hydrolyzed (removing AMP from incorrect aa –> dissociates)
3. editing post-transfer: smaller non-cognate aa are occasionally activated (to form aminoacyl-adenylate) but are transferred to the tRNA aminoacylation site, and then enter the editing site and are hydrylyzed from the tRNA (removing incorrect aa from incorrrectly charged tRNA)

page 443 for figure

Question 4

Ribosomes

structure

Answer 4

2 functionally distinct and separable subunits
large and small
bind separately to mRNA - come apart at end
made of ribosomal RNA and ribosomal proteins
Bacterial ribosome: 70S
- 50s(large)
- 5srRNA, 23s rRNA, ~34 proteins
- 30s (small)
- 16s rRNA, ~21 proteins

Eukaryotic Ribosome: 80S
- 60S (large)
- 5S rRNA, 5.8s rRNA, 28s rRNA, ~49 proteins
- 40S (small)
- 18s rRNA, ~33 proteins

(s is sedimentation speed (greater s = greater sedimentation speed))
most of ribosome is rRNA which has extensive 2ndary and 3ary structure which makes up ribosome structure

role of proteins? primarily to help protect and stabilize structure, probably “interaction points” for regulation

Question 5

Ribosomes

function

Answer 5

basically a platform
- binds mRNA
- localizes tRNA by allowing for codon-anticodon base pairing
- tRNAs do the rest

Question 6

Initiation factors

Answer 6

bact: IF1, IF2, IF3
euks: eIF1-6

Question 7

Elongation Factors

Answer 7

bact:EFG, EFTu, EFTs
euks: eEF1A,B, eEF2

Question 8

Termination Factors

Answer 8

bacteria: RF1, RF2, RF3
Euks: eRF1, ABCE1

Question 9

What two steps in translation are very similar between bact and euks?

Answer 9

elongation, termination

Question 10

IF2

Answer 10

with IF1,IF3 bound, helps tRNA(initiating) bind to small subunit?
G-protein
after association, IF2 hydrolyzes its GTP –> IF1,2,3 dissociate
…large subunit can now bind! ready for elongation

bact

Question 11

IF1,3

Answer 11

helps the small/large subunits, actually kinda keeping them apart
prevent premature association of the large subunit

bact

Question 12

Shine-Dalgarno sequence

Answer 12

ribosome binding site
AGGAGG ~6-9bp~AUG
the purine-rich Shine-Dalgarno seq specifically interacts with a pyrimidine rich region of the 16S rRNA (base pairs with 3’ end of 16s rRNA of small subunit
bacteria and archaea

Question 13

G-proteins

Answer 13

generally have 2 conformational states
protein: GTP –> protein:GDP
active –> inactive
conformational change done by the protein itself

Question 14

initiating tRNA

Answer 14

a special met-containing tRNA
bacteria: formyl-met attached
euks: just a diff sequence in tRNA

Question 15

Marilyn Kozak’s proposal

Answer 15

upstream AUGs would inhibit correct initiation of ribosome in euks

Question 16

Initiation in Euks

Answer 16

1. cap & polyA tail are important to initiation of translation
2. many more IFs than in bact
3. small subunit binds to IFs that are bound to cap & polyA tail
4. then scans in a 5’–>3’ direction down mRNA, eIF2 + tRNAinit bind as it scans
5. stops at 1st AUG
6. eIFs change conformation and leave
7. large subunit joins

Question 16

Initiation in Euks

Answer 16

1. cap & polyA tail are important to initiation of translation
2. many more IFs than in bact
3. small subunit binds to IFs that are bound to cap & polyA tail
4. then scans in a 5’–>3’ direction down mRNA, eIF2 + tRNAinit bind as it scans
5. stops at 1st AUG
6. eIFs change conformation and leave
7. large subunit joins

Question 17

Kozak Sequence

Answer 17

a preffered context around AUG required to stop scanning
RNNAAUGG

in eukaryotes helps w initiation - ribosome can recognize this, acts as a sort of initiation site

R = A or G, N = any

Question 18

eIF1, eIF1A

Answer 18

same role as IF1, IF3
prevent premature association of large subunit

Question 19

eIF5 + eIF2

Answer 19

same job as IF2
with eIF1, eIF1A bound, helps inititating tRNA bind to small subunit
G-proteins

Question 20

eIF3,4,6

Answer 20

involved in cap binding + other roles
eIF4 - really important, lots of subunits/complexes

Question 21

eIF4 subunits

Answer 21

eIF4E, eIF4G bind cap + proteins at polyA tail
eIF4A, eIF4B are RNA helicases

Question 22

GAP

Answer 22

GTPase Activating Protein
“encourages” GTP hydrolysis in G proteins

some of the translocation factors of the ribosome itself acts as GAP or GEF

Question 23

GEF

Answer 23

Guanine Exchange Factor
forces GDP dissociation from inactive protein

some of the translocation factors of the ribosome itself acts as GAP or GEF

Question 24

Elongation basics

Answer 24

1. charged tRNA will base pair with codon now in A site 2. peptide bond will form (no EFs for this) 3. ribosome has to move very similar in bact and euks

Question 25

EFTu

Answer 25

binds tRNA, brings to A site G protein (ribosome changes its conformation to lead to GTP hydrolysis by factor --> leaves also helps ribosome w Kinetic Proofreading (tRNA selection) | bact

Question 26

eEF1A

Answer 26

binds tRNA, brings to A site G protein (ribosome changes its conformation to lead to GTP hydrolysis by factor --> leaves also helps ribosome w Kinetic Proofreading (tRNA selection) | euks

Question 27

EFTs

Answer 27

acts as GEFs for EFTu | bact

Question 28

eEF1B

Answer 28

acts as GEFs for eIF1A | euks

Question 29

EFG

Answer 29

binds to ribosome to promote movement | bact

Question 30

eEF2

Answer 30

binds to ribosome to promote movement also a G-protein, so conformational change that helps the ribosome move is done by GTP hydrolysis in EFG | euks

Question 31

Kinetic Proofreading | tRNA selection

Answer 31

1. initial selection phase: an aminoacyl-tRNA complexed w EFTu can be rejected from the ribosome before EFTu hydrolyzes GTP 2. proofreading phase: following GTP hydrolysis and the departure of EFTu, ribosomes can still reject near-cognate tRNA in this phase both of these are thermodynamic rejection steps

Question 32

Peptide bond formation mechanism

Answer 32

facilitated by 2 loops in the large rRNA that base pair to "C"s in CCA on tRNAs "P loop" --> bp to tRNA in P site "A loop" --> bp to tRNA in A site peptide bond formation itself catalyzed by tRNA 2'OH

Question 33

movement of ribosome mechanism

Answer 33

EFG (eEF2) interacts with ribosome as "tRNA mimic" EFG structure "looks like" EFTu:tRNA - forces its way in, pushes things down

Question 34

Termination Mechanism

Answer 34

no natural tRNAs w anti-codon to the stop codons instead, RFs bind to A site at stop codon these RF are tRNA mimics (like EFG) in bact: RF1, RF2 in euks: eRF1 There are additional RFs that lead to hydrolysis of the tRNA peptide bond (cleave free peptide, break up ribosome) (ABCE1, RF3) = G proteins

Question 35

RF1, RF2

Answer 35

RF1 = recognizes UAA, UAG RF2 = recognizes UAA, UGA

Question 36

eRF1

Answer 36

recognizes all 3 stop codons

Question 37

RF3

Answer 37

in bact, destabilizes complex + leads to peptide hydrolysis from tRNA

Question 38

ABCE1

Answer 38

in euks, destabilizes complex + leads to peptide hydrolysis from tRNA

Question 39

Weird things that can happen during elongation or termination

Answer 39

1. stop codon read through 2. insertion of unusual aa at the stop codon 3. ribosomal frameshifting (programmed)

Question 40

Stop Codon Readthrough | AKA nonsense suppression

Answer 40

there is a low level of stop codon readthrough that occurs naturally because if a tRNA matches partially and if it stays in A site long enough --> peptide bond Result is polypeptide will be longer w/some particular aa where it should've stopped Ex: MMLV (leukemia virus) mRNA --> long polypeptide cleaved by proteases to make 3 gene product (gag-pol-env) ... need much more gag...STOP codon in between gag and pol 1. pseudoknot coded in RNA sequence to pause ribosome 2. pol gene product binds to eRF1 so less likely to terminate

Question 41

Unusual AA: selenocysteine example

Answer 41

found in all 3 kingdoms special tRNA that can be charged with serine by sertRNA synthetase sequence downstream of stop codon "EF" selB that binds charged serTRNAstop + downstream sequence

Question 42

Unusual AA: pyrrolysine example

Answer 42

special tRNA for pyrrolysine + special tRNA synthetase

Question 43

Ribosomal Frameshifting: RF2 gene | bact

Answer 43

its amount is regulated by ribosomal frameshifting one thing impt: "slippery sequence" (tRNA can basepair in 2 frames) if RF2 levels are sufficient, STOP codon in middle of gene gets used (inactive RF2) if RF2 levels are low, ribosome sits at stop, can shift --> new reading frame (active RF2)

Question 44

Ribosomal Frameshifting: RSV | euks

Answer 44

RSV makes gag-pol-env polyprotein by frameshifting 90% of time, stop codon is used (gag only) 10% of time, pseudoknot pauses ribosome, shifts -1, gag-pol-env translated