Lecture 20 Flashcards
Why it’s hard to achieve specificity in aaRS
Use the two very small differences to get the job done right between val and isoleucine
Review: proof reading DNA polymerase: 3-5’ exonuclease
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
Proof reading in tRNA synthetases
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
Proof reading by an aaRS- example
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
Proof reading by an aaRS
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)
Defecting proof reading effects
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
How big is tRNA synthetase?
HUGE- a lot of proteins just to make proteins
ribosome: 2 subunits, one of the biggest things in the cell
Prokaryotes vs eukaryotes pol
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)
Ribosomal RNAs have complicated secondary structures
rRNA of the bacterial small subunit
complicated molecule, lot son secondary structure
this is important for the function of the ribosome
The folding of the ribosomal subunit is highly conserved
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
ribosomal proteins lie mainly on the surface
proteins almost all on the surface
very few where the subunits come together
RNA part also determines how they come together
Distinctive features of the eukaryotic ribosome map to the cytoplasmic surface
most conserved region is surrounding polypeptide exit tunnel- probably b/c this is so important
mRNA with multiple translating ribosomes: a polysome
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
mRNA with multiple translating ribosomes is called a polysome
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
Three stages in translation
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
In bacterial initiation, ribosome small subunit binds directly to shine dalgarno initiation sites on mRNA
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
In eukaryotic initiation, the small subunit binds the 7-methyl-G cap, then scans 5-3’ to find a start codon
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
to understand initiation, you must first understand the basics of elongation
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
Three major steps in elongation
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
Initiation in bacteria
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
Initiation in bacteria
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.
Starting state for elongation:
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
Eukaryotic initiation: the small subunit scans from 5’ cap until it finds a start codon
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
eIF4 complex binds mRNA 5’ cap & poly A tail
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
