Download LECT35 trans1

Protein Biosynthesis
• Away from the Ribosome
Genetic code
Charging tRNA
Ribosomes
• On the Ribosome
Initiation complex
Elongation factors
Peptide bond formation
Termination
Characteristics of the Code
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non-overlapping
degenerate
triplet
basically universal over all living species
polar
no punctuation
subject to miscues
defines a reading frame
Framing the Code
OLD MAN AND THE SEA
Ernest Hemmingway
Garbled
OLD MAA NAN DTH ESE A
Insert A
Garbled
OLD MAA ANA NDT HES EA
Insert A A
In frame
OLD MAA ANA AND THE SEA Insert AAA
N In frame
OLD MAA NAD THE SEA
^
Insert A
delete N
tRNA
Charging tRNA
Q: What is meant by Charging
A: Charging means placing an amino acid on the
3’ (acceptor) end of the tRNA
Q: So, what’s the big deal?
A: There are 20 amino acids; the code is degenerate
There could be 4 “isoaccepting tRNAs” competing for one
Q: I still don’t see a problem
A: One enzyme must recognize 4 different tRNA species
and select the correct amino acid.
Q: One enzyme does all that?
A: No, each tRNA has its own enzyme
Q: What is this enzyme called?
A: Its call Aminoacyl-tRNA Synthetase
Q: So, there are 20 of these enzymes
A: Yes
Q: That makes the job a recognition a little easier then?
A: Yes, but the enzymes still have to distinguish
between look-alikes such as leucine and valine, glutamine
and glutamate, tyrosine and phenylalanine.
Q: Are all aminoacyl-tRNA synthetases alike?
A: Yes and no. Yes, they perform the same function,
i.e., to recognize and transfer the correct amino acid
to tRNA.
Q: Why no?
A: Because one class (Class I) looks for the anticodon on
the tRNA, the other (Class II) looks for other features.
Q: What else?
A: Class I puts the amino acid on the 2’ position of the
terminal ribose on tRNA, Class II only the 3’.
Q: So, how does aminoacyl-tRNA synthetase discriminate
amino acids and different tRNA species?
A: The key lies in the tRNA itself. Besides the anticodon,
tRNAs have other bases that set them apart. These bases
called “identity elements” are found in the terminal ends
(acceptor stem) and internal in the tRNA.
Q: Do they also proofreading?
A: Yes, but sparingly
Q: How sparingly?
A: Enough to keep errors down to isoleucine mistaken
for a valine once every 50,000 times. Ile-tRNA synthetase
actually hydrolyzes the valine-AMP precursor.
CH3
Reaction:
CH3
CH3
CH2 CH-COOCH3
NH3+
L-Valine
CH2-CH2CH-COO- + ATP
NH3+
L-Leucine
PPi
CH3
CH3
O
O
CH2-CH2CH-C ~O-P-O-CH2
O
NH3+
O
Ad
L-Isoleucine
Enzyme Bound
HO
tRNA
OH
CH3
CH2CH-COOCH3CH2
NH3+
Codon-Anticodon Interactions
Polarity
3’
5’
5’ Anticodon on tRNA
3’ Codon on mRNA
3’
5’
Anticodon
loop
mRNA 5’
3rd position
Alanine
C
G
G
C
I
A
(C, U)
Wobble base on
anticodon
3’
mRNAs are always
read 5’ to 3’.
Crevice
Ribosomes: The Staging
Areas of Protein Synthesis
Ribonucleoprotein
Particles
70S (80S) Monosomes
30S (40S)
16S RNA (18S)
23 Peptides (33)
50S (60S)
23S RNA (28S)
31 Peptides (49)
5S RNA (5S + 5.8S)
* Mammalian
tRNA sites
mRNA
30S
50S
Polysomes
Groups of ribosomes attached to a single mRNA