1. Translation
Chapter 9

2. Overview Occurs on ribosomes-large aggregates of rRNA and protein
tRNA acts as amino acid carriers
Prokaryotes—occurs simultaneously with transcription and mRNA degradation
Eukaryotes—occurs in cytoplasm
mRNA translated 5’3’
Protein synthesis aminocarboxy

3. Protein Synthesis
Polymerization of amino acids: condensation reaction (dehydration synthesis)

4. ~Universal Genetic Code
Codons—sets of 3 nucleotides corresponding to a single amino acid
Each codon specifies a single amino acid
More than one codon can specify the same amino acid
code is said to be degenerate
Some aa correspond to a single codon
AUG—initiator codon, methionine (Met, M)
UGG–Tryoptophan (TrP, W)
Often codons encoding the same aa differ onl;y at the 3rd nucleotide
Codons—sets of 3 nucleotides corresponding to a single amino acid
Each codon specifies a single amino acid
More than one codon can specify the same amino acid
code is said to be degenerate
Some aa correspond to a single codon
AUG—initiator codon, methionine (Met, M)
UGG–Tryoptophan (TrP, W)
Often codons encoding the same aa differ onl;y at the 3rd nucleotide

5. ~Universal Genetic Code
The Genetic Code
Chart shows aa assignments
Non-random
Tend to be clustered
Reflects similar codons specifying the same aa
Spontaneous mutations causing a single base change
May not cause an aa change
Similar aa are specified by similar codons
Greatest similarities in first two nucleotides
eg glycine – 4 codons – all GGX
Greatest variability in third nucleotide of the triplet
QUIZ—Codon for R, K, V, F
aa for UCG, CCA, GGG

6. Why~Universal? Exceptions
GUG sometimes used as a start
Mammalian mitochondria
Ciliated protozoa
Selenocysteine
NH3+
COO-
H-C-CH2SeH
GUG sometimes used as a start (bacteria)
Mammalian mitochondria- AUA start, other codons different than consensus. EX UGA-Trp (not Stop)
AGG, AGA-Stop (not Arg)
Ciliated protozoa—Ex UAA and UAG—Gln
Selenocysteine
An essential amino acid for selenoproteins
EX. Glutathione oxidase
Uses unique tRNA, initially bound to Ser
Anticodon recognizes UGA (Stop) as Sel
Signals in 3’ region determine Stop or Sel
Incorporation of selenocysteine by the translational machinery occurs via an interesting and unique mechanism. The tRNA for selenocysteine is charged with serine and then enzymatically selenylated to produce the selenocysteinyl-tRNA. The anticodon of selenocysteinyl-tRNA interacts with a stop codon in the mRNA (UGA) instead of a serine codon. The selenocysteinyl-tRNA has a unique structure that is not recognized by the termination machinery and is brought into the ribosome by a dedicated specific elongation factor. An element in the 3' non-translated region (UTR) of selenoprotein mRNAs determines whether UGA is read as a stop codon or as a selenocysteine codon.

7. Selenocysteine The 21st amino acid?
An essential amino acid for selenoproteins EX. Glutathione oxidase
Uses unique tRNA (tRNASec), initially bound to Ser. Longest known tRNA (95nt).
Anticodon recognizes UGA (Stop) as Sec
Signal (a stem loop configuration) 3’ to the UGA determine Stop or Sec
Dedicated specific elongation factor recognizes the stem-loop and substitutes for usual elongation factor (EF-Tu)
The 21st amino acid?
An essential amino acid for selenoproteins EX. Glutathione oxidase
Uses unique tRNA (tRNASec), initially bound to Ser. Longest known tRNA (95nt).
Anticodon recognizes UGA (Stop) as Sec
Signal (a stem loop configuration) 3’ to the UGA determine Stop or Sec
Dedicated specific elongation factor recognizes the stem-loop and substitutes for usual elongation factor (EF-Tu)
Incorporation of selenocysteine by the translational machinery occurs via an interesting and unique mechanism. The tRNA for selenocysteine is charged with serine and then enzymatically selenylated to produce the selenocysteinyl-tRNA. The anticodon of selenocysteinyl-tRNA interacts with a stop codon in the mRNA (UGA) instead of a serine codon. The selenocysteinyl-tRNA has a unique structure that is not recognized by the termination machinery and is brought into the ribosome by a dedicated specific elongation factor. An element in the 3' non-translated region (UTR) of selenoprotein mRNAs determines whether UGA is read as a stop codon or as a selenocysteine codon.

8. Degeneracy—Wobble Hypothesis
Explains how some tRNA recognize more than one codons
tRNA molecules only need to make strong base pairs with 2 of the three codons in the nucleotide
This third loose base pairing interaction is called wobble
Note: only certain bases can substitute for others
Explains how some tRNA recognize more than one codons
tRNA molecules only need to make strong base pairs with 2 of the three codons in the nucleotide
This third loose base pairing interaction is called wobble
Note: only certain bases can substitute for others
How?
Canonical base pairing with the 1st two codon bases
Loose, weak base pair interactions with the 3rd codon base
#1 nucleotide of the anticodon is in a flexible domain of the tRNA
Why?
Kinetic advantage
tRNA can dissociate more readily from the RNA template
Allows faster protein synthesis

9. Wobble Example This UCA codon was read by the
tRNA with a UGA anticodon
But if this UCA was UCG, it would still have been read by the tRNA with a UGA anticodon
In DNA , no pyr-pyr or pur-pur, but RNA is single stranded, allows more flexibility.
Proposed by Crick (model building). Confirmed by experimentation.
Implications for GE? Primer design

10. Codon Usage
More than one codon exist for most amino acids (except Met and Trp)
Organism may have a preferred codon for a particular amino acid
Codon usage correlates with abundance of tRNAs (preferred codons are represented by abundant tRNAs)
Rare tRNAs correspond to rarely used codons
mRNAs containing rare codons experience slow translation
Implications for GE?

11. Amino Acyl Synthetase and tRNA
Amino acyl synthetases catalyze attachment of aa to its appropriate tRNA
One for each amino acid
tRNA
Derived from large 1º transcript
Heavily modified, unusual bases
Extensive folding due to internal H-bonding
There are 20 amino acids and many tRNAs--over 400 possible combinations between the two. Redundancy in the code means that there are more codons than amino acids.
It is the process of linking the appropriate tRNA to its specific amino acid that is considered the second genetic code.
The specificity of the reaction is governed by tRNA identity elements that are recognized by enzymes called tRNA synthetases.

12. Amino Acyl Synthetase Carboxy end of aa attached to -phospate of ATP
AMP released as carboxy end of amoinoacyl group transferred to O at C-3 of 3’nt
When aa is attached, tRNA is charged or acylated
No aa = uncharged
Wrong aa = mischarged
NOTE PPi
Carboxy end of aa attached to -phospate of ATP
AMP released as carboxy end of aa transferred to O at C-3 of 3’nt
When aa is attached, tRNA is charged or acylated
No aa = uncharged
Wrong aa = mischarged
NOTE PPi

13. tRNA Activation by Aminoacyl tRNA Synthetases
1. Aminoacyl-AMP formation: O HO
(-)O R P O O
O(-) P O O
Adenine +H 3 N C P +
PPi R O O O O O-
Adenine O
O(-) P +H 3 N C O O O- O OH OH
Aminoacyl adenylate
(Aminoacyl-AMP)
2Pi OH OH
2. Aminoacyl transfer to the appropriate tRNA:
The generation of aminoacyl-tRNA's is a two-step process carried out by a family of enzymes called aminoacyl-tRNA synthetases.
1.The amino acid is first activated by conversion of the free carboxylic acid to aminoacyl adenlylate. Although stable, the aminoacyl-AMP intermediate does not leave the enzyme.
2. The amino acid is then transferred to the appropriate tRNA
What happens to PPi?
Because of the pyrophosphatases present in the cell, the pyrophosphate produce (Ppi, inorganic pyrophosphate), is quickly hydrolyzed to Pi, rendering the reaction thermodynamically favorable and irreverisble R R O O O
Adenine +H 3 N C P +
HO-ACC-tRNA +H 3 N C
ACC-tRNA +
AMP O O O- O O OH OH
Overall reaction: amino acid + tRNA + ATP  aminoacyl-tRNA + AMP + PPi

14. tRNA Function and Structure
Anticodon-complementary to codon on mRNA
Amino attachment (CCA) site
Other recognition sites
DHU loop
TC Loop
Extra arm (variable)
NOTE: also unusual bases observed acceptor stem
acceptor stem
Anticodon-complementary to codon on mRNA. NOTE: no normal tRNA complementary to UGA, UAA, or UAG (except tRNSSec)
Amino attachment (CCA) the 3’ end of the tRNA. This is the place at which the specific amino acid is attached
These features generally reside within the acceptor helix, the anticodon stem-loop, and in some systems the variable pocket of the tRNA. In the alanine system, fidelity is ensured by a G·U wobble base pair located at the third position within the acceptor helix of alanine tRNA.

15. tRNA Recognition by Amino Acyl Synthetase
Sequence elements in each tRNA are recognized by its specific synthetase including:
One or more of 3 bases in acceptor stem
Base at position 73 “Discriminator base”
Seems to play a major role in many cases, but in other cases it is completely ignored.
In many, at least one anticodon base
The features that permit recognition are not universal, yet there are some general rules. Recognition is not limited to the anticodon end of the tRNA.
Discriminator base is invariant in every tRNA for a particular amino acid

16. No common set of rules for tRNA recognition !!!
Recognition (cont’d)
No common set of rules for tRNA recognition !!!
Anticodon region is not the only recognition site
The "inside of the L" and other regions of the tRNA molecule are also important
Specificity of several aminoacyl-tRNA synthetases determined by:
one or more bases in anticodon
one or more bases in the acceptor stem
discriminator base 73

17. Mischarging
Observation: several aa similar in size and shape, but mischarging rare.
Editing carried out by aa rRNA synthetase
Ex Double sieve of isoleucine synthetase
Activation site– coarse sieve, rejects aa larger than ile. excluded because they don’t fit.
Editing (hydrolytic) site—fine sieve. Accepts activated amino acids that are smaller than ile (ex, Val-AMP), but rejects Ile-AMP (too large). those that get through are hydrolyzed to aa and AMP. Reduces mischarging from 1/225 (expected) to 1/180,000 (observed).
Sites can also distinguish based on hydrophobicity
There are 20 amino acids and many tRNAs--over 400 possible combinations between the two. Redundancy in the code means that there are more codons than amino acids.
It is the process of linking the appropriate tRNA to its specific amino acid that is considered the second genetic code.
The specificity of the reaction is governed by tRNA identity elements that are recognized by enzymes called tRNA synthetases.
Observation: several aa similar in size and shape, but mischarging rare.
Editing carried out by aa tRNA synthetase
Ex Double sieve of isoleucine synthetase
Expect 1 mischarge/225 events. Consequence? 1 misincorporated aa every time Ile is indicated. Intolerable.
Activation site– coarse sieve, rejects aa larger than ile. excluded because they don’t fit.
Editing (hydrolytic) site—fine sieve. Accepts activated amino acids that are smaller than ile (ex, Val-AMP), but rejects Ile-AMP (too large). those that get through are hydrolyzed to aa and AMP. Reduces mischarging from 1/225 (expected) to 1/180,000 (observed).
Sites can also distinguish based on hydrophobicity

18. Isoleucil-tRNA Synthetase: Proofreading Based on Size
Recognition of aa by tRNA synthetases is based on size and hydrophilicity of aa chainThe recognition of amino acids by aminoacyl-tRNA synthetases is generally based on the size of the amino acid side chain.
 •Acylation site rejects amino acids that are larger than the correct one because the binding site is too small.
•Hydrolytic site destroys activated intermediates that are smaller than the right amino acid.
Example: Valine vs isoleucine (isoleucine has an extra methyl group)
If valine is mistakenly activated by tRNA coding for isoleucine, it is hydrolyzed preventing its incorporation in tRNAIle :
Acylation site rejects aa that are too large to fit in active site. Hydrolytric site destroys activated intermedeated that are smaller than correct aa
Many synthases possses hydrolytic site. If Val is mistakenly activated the Ile tRNA, it will be hydrolyzed by smaller hydrolytic site
Larger
Acylation Site
Smaller
Hydrolytic Site
Larger
Acylation Site
Smaller
Hydrolytic Site CH 3 CH 3 H 3 C CH 3 O O +H 3 N NH 3 + O
tRNAIle O
tRNAIle CH 3 H 3 C CH 3 CH 3 O O +H 3 N +H 3 N O
tRNAIle O
tRNAIle
Ile
Val
Misacylation
Correct Acylation

19. Valyl tRNAVal Synthetase Proofreading: Hydrophobic/Polar Recognition Motif
Acylation and hydrolytic sites can also discriminate based on hydrophobic verses polar interactions.
Example: Valine vs threonine (difference is –OH in place of CH3)
If threonine is mistakenly activated by tRNA coding for valine, it is hydrolyzed preventing its incorporation in tRNAVal :
 Anticodon does not always participate in recognition
Mutational studies have shown that recognition features are relatively simple
Ale – GC pair in tRNA acceptor stem
Aminoacyl-tRNA Synthetases Have two roles
Aminoacyl-tRNA synthetases do the critical job - linking the right amino acid with "cognate" tRNA
They act as a “scaffold” to match up the tRNA with its correct (“cognate”) amino acid
They catalyze a two-step reaction
This generates an ester linkage between
-the 3’OH of the tRNA (on the acceptor stem)
-and the COO- group of the amino acid2) This reaction activates the amino acid for protein synthesisAll members of one set of tRNAs for a particular amino acid (isoacceptor tRNAs) are served by one Aa tRNA synthetase.
Hydrophobic
Acylation Site
Polar
Hydrolytic Site
Hydrophobic
Acylation Site
Polar
Hydrolytic Site 3 HC CH 3 H 3 C OH O O +H 3 N NH 3 + O
tRNAVal
tRNAVal O
Difference in Hydrophobicity
CH3 CH 3 HO CH 3 O O +H 3 N +H 3 N O
tRNAVal O
tRNAVal
Val
Thr
Correct Acylation
Misacylation

20. Experiment (1962) tRNA-ACA Protein has Cys Cys-tRNA-ACA
Anticodon (recognizes UGU codon, encodes Cys)
tRNA-ACA
Cell-free extract amino acids & enymes
tRNA is charged with Cys
Protein has Cys
RNA template
UGUGUGUGUG...
Cys-tRNA-ACA
Treat w metal catalyst removes thiol groups
Charged amino acid is changed chemically
This experiment indicates the amino acid makes no contribution to accurate translation
Protein has Ala
RNA template
UGUGUGUGUG...
Ala-tRNA-ACA
Once an aminoacyl-tRNA has been synthesized the amino acid part makes no contribution to accurate translation of the mRNA.