Essential components of the activation step?
20 aa.'s 20 aminoacyl-tRNA synthetases 32 or more tRNA's ATP MG2+
Steps in protein synthesis?
- Activation of aa.’s
- Initiation
- Elongation
- Termination & ribosome recycling
- Folding and posttranslational processing
Essential components of the Initiation step?
mRNA N-Formylmethionyl-tRNA(fMet) Initiation codon in mRNA (AUG) 30S/50S (bact.) 40S/60S (euk) ribosomal subunits Initiation factors (IF 1, IF2, IF3) GTP MG2+
Essential components of Elongation step?
Functional 70S or 80S ribosome (initiation complex) Aminoacyl-tRNA specified by codons Elongation Factors (EF-Tu, EF-Ts, EF-G) GTP Mg2+
Essential components of Termination and Ribosome recycling?
Termination codon (UAA, UAG, UGA)
Release factors (RF-1, RF-2, RF-3, RRF)
EF-G
IF-3
Essential components of Folding and posttranslational processing?
Specific enzymes, cofactors, other components for removal of initiating residues & signal sequences
Add’l proteolytic processing
Modification of terminal residues and attachment of acetyl, phosphoryl, methyl, carboxyl, carbohydrate or prosthetic groups
Where does attachment of aa. to tRNA occur?
in cytosol (not on ribosome)
What is aminoacylation of tRNA?
attaching proper aa. to proper tRNA using ATP as energy and Mg2+ for stabilization.
once attached the rRNA is “charged”
What must be true of the carboxyl group on the incoming aa. for the new peptide bond to be formed?
The carboxyl group must be activated (phosphate taken from ATP is attached to the carboxyl C) to initiate bond formation
Basic steps in initiation.
- mRNA binds smaller rib. subunit (30S/40S)
- larger rib. subunit binds (50S/60S)
- –> this forms the initiation complex (70S/80S) - initial tRNA base pairs with start codon (AUG, Met)
- GTP is used for energy
- cytosolic initiation factors support the process
Basic steps in Elongation
- New polypeptide lengthened by att. of successive aa’s.
- new aa’s carried to rib. by tRNA
- new aa’s bp with corresp. codon
- elongation factors assist.
- GTP hydrolysis provides e. for tRNA att. and mvmt of pp. along rib.
Basic termination steps.
- Termination codon in mRNA signals end (UAA, UAG, UGA)
- New pp. released from ribosome
- Release Factors assist.
- Rib. recycled for another round
Basic steps of posttranslational processing and folding
- PP fold to proper 3D form
- Enzymatic processing of pp. (before or after folding)
- addn of acetyl, posphoryl, methyl, carboxyl
- att. oligosaccharides or prosthetic groups
Ribosome is made up of what components?
Bacteria: 30S, 50S; 5S, 23S rRNA - note, there is no protein within 18 Å of the active site
final rib. complex = 70S
Eukaryotic: 40S, 60S; rRNA as well
final rib. complex = 80S
What should we really call a ribosome and why?
Ribozyme
most of it is rRNA material
rRNA does the enzymatic work
The mRNA passes ______ the two _______ _________ during translation.
between the two ribosomal subunits
What are the three sites of the ribosomes that bind tRNA’s?
A = aminoacyl site P = peptidyl site E = exit site
What binds to the aminoacyl site?
aminoacyl-tRNA’s
What binds to the peptidyl site?
aminoacyl-tRNA
What binds to the exit site?
uncharged tRNA after it has completed its task on the ribosome
What makes up a tRNA?
single strand of RNA folded into 3D structure
How does a tRNA pick up an aa?
Each tRNA recognizes exactly one aa.
aa. attaches to the amino acid arm
Aminoacyl-tRNA synthetases esterify the aa. to its site on the tRNA
Where does the tRNA pick up its aa?
in the cytosol (not on the ribosome)
What is the “shape” of a tRNA?
in 2D it is a 4-arm cloverleaf.
*aa. arm *anticodon arm *D arm *TpsiC arm (longer have 5th arm)
in 3D is a “twisted L” or gun form
What does the small subunit on the ribosome do/contain?
Contains decoding groove *mRNA’s pass through decoding groove
*tRNA’s enter and read the message in the decoding groove
What does the larger ribosomal subunit contain?
Catalytic Center!!!
*PTC = peptidyl transferase center
*is made of RNA –> ribozyme !!
uses RNA to perform catalysis
Where is the amino acid arm of the tRNA?
at the 3’ end
What is special about the D arm?
contains the unusual nucleotide dihydrouridine (D)
What bond does the aminoacyl-tRNA synthesize?
an ester bond R-C(=O)-OR’ between aa and its tRNA
How many tRNA’s can carry a particular aa?
usually one, sometimes more if the aa has multiple codons
How many aa’s can a tRNA carry?
one
how many aa’s and tRNA’s can an aminoacyl-tRNA synthetase work with?
one aa and one or more tRNA’s (when the aa has multiple codons)
What is the function of the tRNA anticodon loop?
base pairs with the mRNA message codon in the usual complimentary base pairing way
—> wobble pairing at the 3rd codon allows some tRNA’s to pair with more than one codon
What do initiation factors do?
bring the ribosome to the message and assist in assembling the translation machinery
How many initiation factors do bacteria have?
How many initiation factors do eukaryotes have?
Bact: 3 IF1, IF2, IF3
Euk: over a dozen…
What do elongation factors do?
Deliver the tRNA’s and move the ribosome down the message
What do termination / recycling factors do?
end translation at a stop codon (UAA, UAG, UGA)
dissociate the subunits for reuse
What is the reading frame?
the start codon AUG sets the reading frame (codon = 3 nucleotides long).
there is no overlap from codon to codon
When/where is the reading frame established?
during initiation when the machinery is being established at the start codon (AUG, Met)
To begin initiation, the _________ and ________ must assemble in the ________ of the ribosome.
start codon (5’AUG)
Met-tRNA
P-site
The next aa. in the growing chain arrives at the __________ before it enters the ________ site.
A site
P site
What is the direction of the synthesis of the growing pp chain?
N —> C
What is the residue at the Amino Terminal of every new pp?
Met (5’AUG)
How many tRNA’s does bacteria have for Methionine?
Two.
One is used for the 5’AUG start codon (fMet).
One is used for an internal AUG methionine code.
Methionine side chain?
-CH2-CH2-SH-CH3
How many tRNA’s does eukaryotic cells have for Methionine?
One
regular tRNA(Met)
During elongation, where does the incoming tRNA bind?
First to the A site, then to the P and E sites.
only the first tRNA(Met) binds directly to the P site
The GTP that provides the energy for the initiating pp. bond is attached to _________.
IF2
In eukaryotes ________ is required to bind the 7-methylguanosine cap at the _________ end of the __________.
eIF 4E
5’
mRNA
What other eIF’s are assembled during euk. initiation?
4G, 4A, 4B, etc.
In eukaryotes, how does the ribosome find the start codon?
scans to find AUG (Met)
What are IRES’s?
Internal ribosomal entry sites.
often exploited by viruses after shutting down the host cell cap-dependent synthesis
What step is considered to be the most controlled/regulated in translation?
initiation
During elongation, what delivers the inconing aa-tRNA to the A site?
Euk: EF1A
Bact: EF-Tu
How does termination begin?
Recycling Factor reads stop codon (UAA, UAG or UGA) in the A-site
RF moves to tRNA’s space and terminates peptide chain
How does termination end?
Release of pp chain
dissociation of subunits
protein goes off to fold, receive modifications
What molecule is the decoding molecule?
tRNA
How many possible codons are possible?
4^3 = 64
yay, math!
What is degeneracy in regards to codons?
multiple 3-nucleotide codons for a single aa.
How many aa’s are specified by just the first two nucleotides in the codon?
6 aa. where just the first two nucleotides matter.
In general, are charged tRNA’s involved in regulation of translation?
No. There are generally enough of the around.
If the wrong aa. is put on a tRNA, how is this “proofread”?
- The aminoacyl-tRNA synthetase has a built-in proofreading mechanism that we don’t understand very well. (they are diff. for diff. synthetases)
- The elongation factor that brings the charged tRNA to the rib. won’t bind very well.
Where did the names for the ribosomal subunits come from?
30S / 50S / 70S
40S / 60S / 80S
sedimentation rates through a centrifuge
What happens at the A site?
Charged tRNA’s enter into the ribosome
What happens at the P site?
tRNA gives up its aa to the growing pp chain.
What happens at the E site?
uncharged tRNA slides here then exits
During bacterial initiation of translation, first _________ and ________ bind to the _________ subunit.
IF1 and IF3
30S
Once IF1 and IF3 are bound to 30S, _________ binds to ________ using ________ as a guide.
mRNA
30S
Shine-Dalgaro
Once mRNA is bound to the 30S subunit at initiation, _________ brings a special ________ to the ________ to pair with the _______codon.
IF2
fMet-tRNA (bacteria only)
P-site
AUG
Hydrolysis of ________ bound to ________ provides energy to release initiation factors and binding of ___________.
GTP
IF2
50S subunit
What is special about the Shine-Delgardo sequence?
is how ribosome recognizes the correct AUG start codon.
is upstream form AUG
Watson-Crick base pairs with 3’ end of rRNA within small subunit
will position AUG correctly in the P-site
During elongation, peptide bond formation is catalyzed by _______ in the __________.
Peptidyl Transferase in the
Peptidyl Transferase Center (PTC)
In eukaryotes, scanning for AUG start codon, scanning is _________ dependent and is more ________.
ATP
regulated
In eukaryotes, what elongation factor ________ brings charged ________ to the vacant _______ site.
EF1 A
tRNA’s
A
After a peptide bond is formed, _______ and _______ hydrolysis triggers movement of the _________ and ________ one codon in the 3’ direction.
EF2
GTP
mRNA and tRNA
Where does the energy for peptide bond formation come from?
The ATP used in tRNA charging.
Formation of one peptide bond requires _________ high energy bonds
4
What are the high energy bonds used to form a peptide bond?
- Charge tRNA: 2 (ATP —-> AMP)
- Deliver aa-tRNA to A site: 1 (GTP —> GDP)
- Translocation: 1 (GTP —> GDP)
