Proteins and Such for Exam 4 Flashcards
tRNAs
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)
tRNA synthetases
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
tRNA synthetase “editing”
- size exclusion (not rlly editing): large non-cognate aa are exclude from the aminoacylation site
- 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)
- 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
Ribosomes
structure
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
Ribosomes
function
basically a platform
- binds mRNA
- localizes tRNA by allowing for codon-anticodon base pairing
- tRNAs do the rest
Initiation factors
bact: IF1, IF2, IF3
euks: eIF1-6
Elongation Factors
bact:EFG, EFTu, EFTs
euks: eEF1A,B, eEF2
Termination Factors
bacteria: RF1, RF2, RF3
Euks: eRF1, ABCE1
What two steps in translation are very similar between bact and euks?
elongation, termination
IF2
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
IF1,3
helps the small/large subunits, actually kinda keeping them apart
prevent premature association of the large subunit
bact
Shine-Dalgarno sequence
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
G-proteins
generally have 2 conformational states
protein: GTP –> protein:GDP
active –> inactive
conformational change done by the protein itself
initiating tRNA
a special met-containing tRNA
bacteria: formyl-met attached
euks: just a diff sequence in tRNA
Marilyn Kozak’s proposal
upstream AUGs would inhibit correct initiation of ribosome in euks
Initiation in Euks
- cap & polyA tail are important to initiation of translation
- many more IFs than in bact
- small subunit binds to IFs that are bound to cap & polyA tail
- then scans in a 5’–>3’ direction down mRNA, eIF2 + tRNAinit bind as it scans
- stops at 1st AUG
- eIFs change conformation and leave
- large subunit joins
Initiation in Euks
- cap & polyA tail are important to initiation of translation
- many more IFs than in bact
- small subunit binds to IFs that are bound to cap & polyA tail
- then scans in a 5’–>3’ direction down mRNA, eIF2 + tRNAinit bind as it scans
- stops at 1st AUG
- eIFs change conformation and leave
- large subunit joins
Kozak Sequence
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
eIF1, eIF1A
same role as IF1, IF3
prevent premature association of large subunit
eIF5 + eIF2
same job as IF2
with eIF1, eIF1A bound, helps inititating tRNA bind to small subunit
G-proteins
eIF3,4,6
involved in cap binding + other roles
eIF4 - really important, lots of subunits/complexes
eIF4 subunits
eIF4E, eIF4G bind cap + proteins at polyA tail
eIF4A, eIF4B are RNA helicases
GAP
GTPase Activating Protein
“encourages” GTP hydrolysis in G proteins
some of the translocation factors of the ribosome itself acts as GAP or GEF
GEF
Guanine Exchange Factor
forces GDP dissociation from inactive protein
some of the translocation factors of the ribosome itself acts as GAP or GEF
Elongation basics
- charged tRNA will base pair with codon now in A site
- peptide bond will form (no EFs for this)
- ribosome has to move
very similar in bact and euks
EFTu
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
eEF1A
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
EFTs
acts as GEFs for EFTu
bact
eEF1B
acts as GEFs for eIF1A
euks
EFG
binds to ribosome to promote movement
bact
eEF2
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
Kinetic Proofreading
tRNA selection
- initial selection phase: an aminoacyl-tRNA complexed w EFTu can be rejected from the ribosome before EFTu hydrolyzes GTP
- 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
Peptide bond formation mechanism
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
movement of ribosome mechanism
EFG (eEF2) interacts with ribosome as “tRNA mimic”
EFG structure “looks like” EFTu:tRNA
- forces its way in, pushes things down
Termination Mechanism
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
RF1, RF2
RF1 = recognizes UAA, UAG
RF2 = recognizes UAA, UGA
eRF1
recognizes all 3 stop codons
RF3
in bact, destabilizes complex + leads to peptide hydrolysis from tRNA
ABCE1
in euks, destabilizes complex + leads to peptide hydrolysis from tRNA
Weird things that can happen during elongation or termination
- stop codon read through
- insertion of unusual aa at the stop codon
- ribosomal frameshifting (programmed)
Stop Codon Readthrough
AKA nonsense suppression
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
Unusual AA: selenocysteine example
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
Unusual AA: pyrrolysine example
special tRNA for pyrrolysine + special tRNA synthetase
Ribosomal Frameshifting: RF2 gene
bact
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)
Ribosomal Frameshifting: RSV
euks
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
