Lecture 20

Question 1

Why it’s hard to achieve specificity in aaRS

Answer 1

Use the two very small differences to get the job done right between val and isoleucine

Question 2

Review: proof reading DNA polymerase: 3-5’ exonuclease

Answer 2

similar proof reading in DNA pol
2 sites- catalysis happens in separate site
don’t communicate, but if something swings over it’s probably the wrong base
happened to get over, kinetics allow, would cleave it off
incorrect nucleotide will be removed

Question 3

Proof reading in tRNA synthetases

Answer 3

2 sites
1 catalysis
1 editting
put wrong one on either during
adenylation of amino acid or putting onto trna (charging tRNA)
if something is not stable, something will allow it to be cut off at editing site

Question 4

Proof reading by an aaRS- example

Answer 4

ATP and time are consumed in futile cycle to increase accuracy
case: isoleucine to valine, but there are more cases
normally: valine gets in, gets adenylated
make valine adenylate, chops off
never adds valine adenylate, just cleaves off, end up with val and AMP
val is smaller than isoleucine
only valine will get in
if it has the correct one, will cleave and the correct one won’t be cleaved
steric considerations of size differences of two amino acids
ser and thr have polar hydroxyl groups- could be something there
could do this job just by having steric considerations at synthesis site
worth the extra energy

Question 5

Proof reading by an aaRS

Answer 5

some proofread the amino acyl adenylate
other proof read the aa tRNA
and some don’t bother to proof read at all
(try doesn’t, no similar structures so it won’t make mistakes)

Question 6

Defecting proof reading effects

Answer 6

causes protein misfolding and neurodegeneration
if you don’t do proof reading this is what happens
basis of this disease- mutation in specific aa in editing site of tRNA synthetase
will just make errors at a higher rate
neurodegeneration similar to to huntington’s
invest extra energy to get job done right

Question 7

How big is tRNA synthetase?

Answer 7

HUGE- a lot of proteins just to make proteins

ribosome: 2 subunits, one of the biggest things in the cell

Question 8

Prokaryotes vs eukaryotes pol

Answer 8

2 subunits in both
small one does the job of decoding mRNA
large one does the job of catalyzing peptide bond synthesis
30 and 50 s are the numbers you want to think about
polymerases composed of both ribosomal RNAs and also proteins. euks more complicated than prok. macromolecules made of both proteins and RNA (ribonucleoproteins)

Question 9

Ribosomal RNAs have complicated secondary structures

Answer 9

rRNA of the bacterial small subunit
complicated molecule, lot son secondary structure
this is important for the function of the ribosome

Question 10

The folding of the ribosomal subunit is highly conserved

Answer 10

small and large come together
eukaryotic and prokaryotic are similar, euk maybe has a few extra loops but they are basically the same
b/c such a fundamental process of life
differences enable us to make antibiotics
especially in a fast growing cell
RNA parts are most important. first ribosomes could have only been made out of RNA, figure out that would have had to be the RNA part that was most important. Looking at structure, RNA part makes up most of core shape.
RNA part giving shape, most critical for function, proteins extra/tuning, not essential

Question 11

ribosomal proteins lie mainly on the surface

Answer 11

proteins almost all on the surface
very few where the subunits come together
RNA part also determines how they come together

Question 12

Distinctive features of the eukaryotic ribosome map to the cytoplasmic surface

Answer 12

most conserved region is surrounding polypeptide exit tunnel- probably b/c this is so important

Question 13

mRNA with multiple translating ribosomes: a polysome

Answer 13

synthesis is not with a single ribosome sitting on RNA, but actually a whole bunch
called polysomes
translation goes from the long thin part to the thick short part (double check diagram)
little proteins coming out where you’re starting, as you go along, more protein coming out

Question 14

mRNA with multiple translating ribosomes is called a polysome

Answer 14

where the start codon is is where it starts
5’ end
ribosome sitting there, RNA moving in opp direction
many ribosomes on message allows to make more protein than if only one at a time
protein making is a lot slower than DAN or RNA pol
many ribosomes increases the overall rate
speed is important especially for rapidly dividing cells

Question 15

Three stages in translation

Answer 15

Initiation: the ribosome is placed on the start codon
Elongation: mRNA templated polypeptide polymerization
Termination: the polypeptide and mRNA are released
Note: in eukaryotes, transcription occurs in the nucleus and translation occurs in the cytoplasm. but the directions are the same in all cases. cartoon on slide applies to bacteria
transcription moving left to right
translation moving 5 to 3’ along message, also left to right
same direction
in bacteria: everything happens in the same compartment
replicate chromosome a second time before they’ve even divided
bacteria couple transcription to translation
same time, ribosomes load on and start doing translation

Question 16

In bacterial initiation, ribosome small subunit binds directly to shine dalgarno initiation sites on mRNA

Answer 16

Shine delgarno sequences are upstream of initiation codon
works as ribosome binding site b/c its sequence is complementary to part of small subunits ribosomal rna
small subunit will bind to message in absence of full ribosome

Question 17

In eukaryotic initiation, the small subunit binds the 7-methyl-G cap, then scans 5-3’ to find a start codon

Answer 17

don’t make multiple proteins from the same RNA
in bacteria- how they tell where to start translation
make modifications to rna
5’ methylguanasine cap
protect end, also recognized by small subunit of rna
cruise down once bound and find aug
use a lot of energy

Question 18

to understand initiation, you must first understand the basics of elongation

Answer 18

the ribosome has three binding sites
aminoacyl tRNA
peptidyl tRNA
exit
A P and E sites
all sites where specific tRNAs are gonna be bound
big job: which tRNA is right? that’s what ape decides
select which one is correct for a codon
direction of tRNA and mRNA movement through ribosome is from a site to e site
a is closest to 5’ end
large ribosomal unit has half of each site
small one has other half, plus mRNA binding site

Question 19

Three major steps in elongation

Answer 19

A site: tRNA selection
P site: peptidyl transfer
translocation: uncharged tRNA exits from e site
note that the growing polypeptide chain is transferred onto the incoming aa tRNA
The aa on the incoming aa tRNA is not transferred onto the chain

chain already at that p site (several aa attached to tRNA at P site)
a site open. incoming amino tRNA comes in. bp with whatever codon is at that site. this bping is critical for the selection of the tRNA selection step at a site

reaction where you transfer protein chain. entire protein chain moves from p site to a site. happens at p site, what is catalyzed by large subunit of ribosome
then get empty one to float off from e site, get empty a site, ready for new one

Question 20

Initiation in bacteria

Answer 20

Ingredients:
mRNA
fmet-trNA
initiation factors: IF 1 2 and 3
GTP and Mg2+
Small (30 S) subunit
initially just the small subunit of the RNA
a and p sites are half sites
tRNAs bound to those but you don’t want tRNAs bound to those yet
other initiation factors bind to small subunit to prevent large subunit from coming in and binding until whole thing is ready to go
small sunlit with initiation factors, can bind mRNA at this point but they don’t need to bind mRNA until after they have tRNA there
ultimately: rna set up so codon will be over at p site
IF-1 occupies the small subunit’s A site
AUG start codon aligned in the P site
Shine delgarno sequence binds to small subunit RNA

Question 21

Initiation in bacteria

Answer 21

Bacteria use a specialized initiator tRNA charged with a modified amino acid, fMet
Eukaryotes use plain old met
the presence of peptides containing N terminal fMet is interpreted by animal immune systems as a sign that bacteria are present or that mitochondria have ruptured. in other words, for us fMet is a danger signal
always start with methionine
IF2 bound to gtp will bind to initiation tRNA with fMet bound
initiation factor with this bound will bind to P site of small subunit. will bind without message just by fit. bp not driving binding
fmet binds to initiation factor called IF2 (GTP bound), many of these initiation factors and elongation factors are gtp binding proteins
can use hydrolysis for several things; switch, signal , energy, etc.

Question 22

Starting state for elongation:

Answer 22

Initiation factors have fallen off
large 50s subunit bound
fMet tRNA and AUG codon are in P site
A and E sites empty
uses gtp hydrolysis as a signal to say get going
causes above things to happen
form the complex
tRNA with amino acid, occupying p site, nothing at a or e site
fMet specialized to just go to P site without having to go to the A site first
Mg important for gtp hydrolysis

Question 23

Eukaryotic initiation: the small subunit scans from 5’ cap until it finds a start codon

Answer 23

As a consequence, eukaryotic mRNAs are almost always monocistronic: they contain only a single initiation site and encode only one polypeptide
cistron is a fancy name for a protein coding gene
In some cases, a polyprotein can be cleaved by site-specific proteases to yield more than one polypeptide
This takes energy- euks scan to find AUG
polycistronic: make several from the same thing
euks: only one, but can cleave

Question 24

eIF4 complex binds mRNA 5’ cap & poly A tail

Answer 24

Euks: take s5’ cap and 3’ tail and circularize
might enable to have ribosomes load on, find aug, translate, and then be at the end
jump back on and not waste as much energy trying to find aug
still an area of research

Question 25

Elongation cycle: the hybrid state model of elongation

Answer 25

Selection/accomodation
peptidyl transfer
translocation
each one of these sites is a hybrid between small and large subunit
growing chain at p site, a site empty, throw out what’s in e site
amino acyl tRNA bound to EF Tu GTP binding protein
together bind a site
initially, can’t fully occupy site; bound but has not occupied. bent, must undergo conformational change to move fully into site (accommodation) a proof reading step to make sure you selected the right amino acyl tRNA
once tRNA with aa at a site, growing chain at p site, transfer whole chain from p to a, head stuck in p site and tail stuck in a site still
another elongation factor comes in called EFG
takes energy
gtp binding protein, uses gtp hydrolysis for energy, motor to cause movement and have whole thing slide down a unit
whole thing
back to selection
and accommodation
aa-tRNA: EF-Tu: GTP
binds into a site of ribosome

Question 26

Elongation: aa tRNA selection

Answer 26

The antibiotic tetracycline binds the 30s subunit’s a site, blocking entry of the Aa-tRNA-ef-Tu complex
notice: there are two steps at which the aa tRNA can dissociate (fall off) before peptidyl transfer
this is a true proof reading mechanism
incorrectly base paired tRNAs preferentially dissociate
will bind but only use gtp hydrolysis after stably bound
proof reading mech: 2 steps
1st, get job done right- pick up right base pair. 3nd: independent step to check correct
initial bp with gtp bound, once stably associated, hydrolyze gtp (on a timer)
causes large conformational change, will cause to fully move into that a site
not stable binding: likely to fall off
wrong ones are going to fly in and bounce off
if they stay long enough for hydrolysis to occur, can help move into site
if it doesn’t fit right, will be lost
this proof reading mech helps make it accurate
10^-4, similar to rna
EF Tu most abundant protein in fast growing cell
don’t have it free, all bound up to EF tu, that’s how important it is to get it done right

Question 27

aa-tRNA: EF-Tu: GTP

Answer 27

adopts a bent conformation in the decoding site

will only move in after gtp hydrolysis

Question 28

Kinetic proof reading: the timer function of EF-Tu is critical for accuracy

Answer 28

a slowly hydrolyzed gtp analog increases accuracy of translation but at the expense of speed
used gtp analog
3phos phos
had S
can’t hydrolyze off third phosphate (non hydrolyzable form of gtp)
protein synth increases in accuracy, but slowed down- trade off
might not do as well on rest of the “test”
don’t hydrolyze gtp, longer to check that you have selected the right thing
cost: slow down

Question 29

Elongation: peptidyl transfer

Answer 29

NH2 has free pair of e that can do nucleophilic attack
current thinking: not traditional acid base chemistry, all about entropy. all about orienting substrates and excluding water so reaction is driven by entropy
aa attached to end of tRNA sitting on a site and p site, have nuc attack by amino group in a site onto carbonyl in p site, catalyze transfer

Question 30

Elongation: puromycin is a chain terminator

Answer 30

The antibiotic puromycin mimics an aminoacylated tRNA
but it is a polypeptide chain terminator
antibiotics that target every aspect of protein synthesis
close enough to looking like an aa that it can do attack
no tRNA, nothing to do next step
chain terminator like dideoxy
how they figured out if it’s rna or protein

Question 31

Fragment reaction mimics peptidyl transfer

Answer 31

in tube: peptide transfer
catalyzed this reaction in test tube
revealed: peptidyl transferase enzyme is RNA not protein
killed protein activity, still worked, rna activity did not work