Lecture 35: Protein Synthesis Flashcards
size of bacteria ribosome
70S ribosome
50S and 30S subunits
eukarotic ribosome
80S ribosome
40S and 60S subunits
protein synth starts at
AUG codon (Met)
bacteria start site recognition
Shine Delgarno Sequence
complementary sequence to 16S rRNA before the AUG
eukaryotic start site recognition
scan for 5’ AUG
peptidyl transfer rxn
catalyzed by 23S (28S) rRNA which acts as a ribozyme
amino acid charged tRNA molec binding overview…
bind to A site
catalysis in P site
uncharged tRNA exits from E site
Why GTP hydrolysis??
to move the ribosome
what is a ribozyme
an enzyme made of RNA
what do ribosomes do?
protein synthesis!!!
Bacterial protein synth
happens at same time as RNA synth!!!
RNA pol and ribosomes work together
Many ribosomes can work at the same time?
eukaryotic protein synth
happens in CYTOSOL
MANY ribosomes translating the PROCESSED mRNA transcript at the same time (polyribosome complex)
Protein synth starts….
at 5’ end of mRNA with first amino acid in Nterminus of polypeptide
ribosome structure
large and small ribosomal subunits
contains 3 tRNA sites covering 3 codons in the mRNA
A site
aminocyl
entry site for charged tRNA
P site
peptidyl
contains catalytic residues in rRNA
E site
exit
where uncharged tRNA is expelled from ribosome
at what sites does base pairing between anticodon of tRNA and codon of mRNA take place??
A and P sites of ribosome ONLY
size of ribosomal subuints… molecular terms and Svedberg units
sedimentation rate
bacterial ribosome
several RNA molecs
35+ proteins
large subunit is 50S
total: 70S
eukaryotic ribosome
parge subunit is 60S
total 80S
35+ proteins
Why is the S value for the bacterial ribosome (70S) smaller than the sum of the S values for the 2 subunits (50S ad 30S)
its not simply additive
sedimentation is more than just mass
also takes shape into consideration when associated, the molec seems smaller and more compact than the 2 indiv sub units
what is the catalyst for the peptidyl trasferase rxn?
rRNA
does a ribosome without proteins work?
not well, but it does work a little
one codon for Met
5’AUG3’
Shine Dalgarno sequence
in bacterial mRNA
upstream of initiator Met
forms base pair contacts with 16S rRNA molec in small ribosomal subunit
purine rich
if it isn’t the Shine-Dalgarno doesnt match the 16S rRNA sequence, it will still bind, just with lower affinity
Eukaroytic initiaition and AUG
NO Shine Dalgarno sequence
just can back and forth for the AUG closes to the 5’ end of the mRNA
Formyl Methionine
in bacteria
initiator Met codon is recognized by special tRNA^fMet
first charged w/ Met by aminoacyl synthetase
then a.a. is formylated by rxn with transformulase to get initiator fMet-tRNA^fMet
tRNA charged w/ formyl Met…
is only used at the initial Met codon
Met codons (AUG) within the open reading frame (not initial codon) uses what tRNA? (bacteria)
tRNA^Met
NOT forymlated one
(byt still charged by same aminoacyl synthetase enzyme)
overall bacterial protein synth initiation
step 1
formation of complex containing 30S ribosomal subunit and 2 initiator proteins (IF-1 and IF-3)
then bind to mRNA transcript at initiator codon and Shine Dalgarno seq
overall bacterial protein synth initiation
step 2
complex recognized by GTP bound IF-2
facilitates binding of fMet-tRNA^fMet to AUG codon in P site of ribosome
overall bacterial protein synth initiation
step 3
50S subunit binds and hydrolyzes GTP
release of all IF proteins
final product: 70S initiatior complex
Eukaryotic initiation doesnt…
don’t use shine dalgarno or formyl Met
Eukaryotic initiation
forms iniatior complex w. small ribosoma subunit (40S), eIF2, eIF4E, eIF4G, and PAB INCLUDES 5’ cap!, AUG codon, and PolyAtail at 3’ end
forms a LOOP
this gets recognized by large ribosomal subunit (60S)
scan for AUG start codon (5’ to 3’ direction)
start synth
What mught be the advatage of using this type of “generic” protein complx for initation rather than a “Shine-Dalgarno” ribosome binding site in the 5’ end of the mRNA transcript?
more potential products!
another way of generating different products from the same RNA
you could miss the first AUG and star with the next one
elongation in bacteria: Step 1
binding of GTP bound elongation factor
Ef-Tu faciliates DELIVERY of charged tRNA to A site after GTP hydrolysis
different because A site (not P)!!!
reload EF-Tu elong factor with another GTP to keep elongation going
whats elongation
addition of next a.a. to nascent polypep chain
when is the only time we use the P site
only binding of initator codon is centered on P site
Step 2 of elongation
23S ribozyme catalyzes peptide bond formation
through rxn that is driven by proximity and orientation of substrates (2 a.a.s)
FORMATION OF PEPTIDE BOND dipeptide attached to second tRNA
conf change in tRNAs, but base pairing stays same
Step 3 of elongation
conf change in ribosomal complex
binding of GTP bound elong factor (EF-G) (uses nrg of GTP hydrolysis)
translocate ribosome in 3’ direction
everything moved over one site
A is open, stuff is in T and P, ready for new stuff in A
net effect of elongation steps
net effect: A site is now centered on NEXT codon
dipeptidyl-tRNA centered in P site
there is room for next incoming charged tRNA
Where will the 3rd amino acid end up?
on the BOTTOM
amino acid terminus is formed first, we add to the BOTTOM
Termination
one of the 3 termination codons appears in A site
GTP independent binding of terminator proteins (RELEASE FACTORS (RF)
Release factors
promote hydrolysis and release of polypeptide
then they dissociate
binding of EF-G after RF binding
binding of next EF-G and ribosmome recycling factor (RRF) causes dissociation of ribosome
IF-3 binds to 30S to get readu for another round
dissaemblation of the ribosomal complex
as upstream ribosome on mRNA transcripts approchaes SAME termination codon
electron micrograph slide 17
electron micrograph slide 17
Beginning with uncharged tRNA^AA1 and free AA, how many ATP equivalents are required to synth and release bacterial polypeptide of 50 aas (assume 1GTP=1ATP)
200 ATP equivalents!
See slide 18!
6 steps
but are there usually only 50 aas?
more lie 500…
2000 ATPs needed
Up to 90% of all of the chem nrg used in a cell is dedicatd to proten synth, which means it needs to be TIGHTLY REGULATED. why do you expect that the majority of proten synth regulation is at the level of DNA transcription NOT RNA translation
if we don’t need the product, don’t even start the process at making RNA. its a waste!
how lots of antibiotics work
inhibiting protein synthesis
targer prokaryotic ribosomes
puromycin
looks like 3’ end of charged tRNA
can bind to A site of ribosomal complex
polypeptide chain attached to the antibiotic (by peptidyl transferase rxn)
result: abortibe complex formation
tetracycline
binds to A site
blocks binding of actual charged tRNA molec
effect: stalling ribosome on mRNA trasncript
chloramphenicol
blocks formation of peptide bond
erytheromycin
prevents ribosomal translocation
steptomycin
interferes with codon base pairing
the first antibiotic produced for wide-spread clinical use was penicilin, what is its mode of action?
stops synthesis of cell walls in bacteria
not targeted toward ribosomes
