Transcript document

Chapter 32
The “2nd Genetic Code”
Pages 1075 to 1086
Learning objectives: Understand the following
• The Raney nickel experiment
• The class differences of aminoacyl tRNA synthetases?
• How aminoacyl tRNA synthetases recognize their cognate tRNA
• Crick’s
Wobble
Hypothesis
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tRNAs are bifunctional
specific amino acid
Phe
Acceptor stem
Anticodon loop
AAA
UUU
Codon in mRNA
•Amino acids must be
activated for translation
•Via covalent linkage of
an amino acid to the
3’OH of the tRNA
•This generates a
“charged tRNA” a.k.a.
aminoacyl-tRNA
tRNA activation must be specific
•When a ribosome pairs a "CGC" tRNA with
"GCG"codon, it expects to find an alanine carried by the
tRNA. It has no way of checking.
•This is because codons in the mRNA are recognized by
the anti-codon loop in the aminoacyl-tRNAs
•The delivery of the amino acid is specified by this
codon-anticodon interaction (regardless of which amino
acid is attached to the tRNA)
HOW DO WE KNOW THIS???
From:http://www.rcsb.org/pdb/molecules/pdb16_1.html
Experiment (1962)
tRNA-ACA
Cell-free extract
amino acids & enymes
tRNA is charged with
Cys
Cys-tRNA-ACA
Treat w metal catalyst
removes thiol groups
(Raney nickel)
Anticodon (recognizes
UGU codon, encodes Cys)
RNA template
UGUGUGUGUG...
Charged amino acid is
changed chemically
Ala-tRNA-ACA
Protein
has Cys
Experiment (1962)
tRNA-ACA
Cell-free extract
amino acids & enymes
tRNA is charged with
Cys
Cys-tRNA-ACA
Treat w metal catalyst
removes thiol groups
Anticodon (recognizes
UGU codon, encodes Cys)
RNA template
UGUGUGUGUG...
Protein
has Cys
Charged amino acid is
changed chemically
Ala-tRNA-ACA
RNA template
UGUGUGUGUG...
Protein
has Ala
Once an aminoacyl-tRNA has been synthesized the amino acid
part makes no contribution to accurate translation of the mRNA.
tRNA activation must be specific
•The delivery of the amino acid is specified by this
codon-anticodon interaction (regardless of which amino
acid is attached to the tRNA)
•Each tRNA is matched with its amino acid long before it
reaches the ribosome.
•The match is made by a collection of remarkable
enzymes, the aminoacyl-tRNA synthetases.
•These enzymes charge each tRNA with the proper
amino acid, thus allowing each tRNA to make the proper
translation from the genetic code of DNA into the amino
acid code of proteins.
From:http://www.rcsb.org/pdb/molecules/pdb16_1.html
Aminoacyl-tRNA Synthetases
Have two roles
1) 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 acid
2) This reaction activates the amino acid for protein
synthesis
The Aminoacyl-tRNA Synthetase
Reaction
• The goal of this reaction is to activate an amino
acid by forming an ester linkage with the correct
tRNA
O
Adenine
O-P-OCH2
O
-
O
O
OH
H
CC
3’-most ntd
from the CCA
acceptor stem
OH
C
- C - R group
NH3+
Amino acid
The Aminoacyl-tRNA Synthetase
Reaction is two steps
1) Activate the amino acid first, by reacting with ATP
Amino acid + ATP
Aminoacyl AMP + PPi
2Pi
An enzyme-bound intermediate
2) Transfer the activated amino acid to its cognate
tRNA
Aminoacyl AMP +tRNA
Aminoacyl-tRNA + AMP
The Aminoacyl-tRNA Synthetase
Reaction
• The goal of this reaction is to activate an amino
acid by forming an ester linkage with the correct
tRNA
The Aminoacyl-tRNA Synthetase
Reaction - Step 1
All aminoacyl tRNA synthetase enzymes have this
step in common
Step 2 - differs depending on the enzyme
Transfer of the amino
acid to the 2’OH of the
tRNA first, then the 3’OH
Transfer of the amino
acid to the 3’OH of the
tRNA
Aminoacyl-tRNA Synthetases
• Despite their common function, the
synthetases are a very diverse collection of
enzymes
• Four different quaternary structures: , 2, 4
and 22
• The subunits vary in size from 334 to more
than 1000 amino acids
• Two different reaction mechanisms (as seen
on previous slide)
There are at least 20 different AminoacyltRNA Synthetases
How do we name them?
tRNAAla + Ala
Ala-tRNAAla
Enzyme = alanyl tRNA synthetase
• Are grouped into Class I or Class II based on:
1) monomers or oligomers
2) type of reaction mechanism
3) general features of their amino acid substrate
4) short stretches of amino acid similarity
The two Classes of
Aminoacyl-tRNA Synthetases
• Larger, more
hydrophobic
amino acid
substrates
• Class I
enzymes are
monomers
Class I
Class II
Arg
Cys
Gln
Glu
Ile
Leu
Met
Trp
Tyr
Val
Ala
Asn
Asp
Gly
His
Lys
Phe
Pro
Ser
Thr
• Smaller, more
hydrophilic
amino acid
substrates
• Class II
enzymes are
multimers
Different Structures of
Aminoacyl-tRNA Synthetases
•The one shown in the next slide,which charges
aspartic acid onto the proper tRNA, is a dimer of
two identical subunits (colored blue and green,
the two tRNA molecules are colored red).
•Others are small monomers or large monomers,
or dimers, or even tetramers of one or more
different types of subunits.
•Some have wildly exotic shapes, such as the
serine enzyme.
Aspartyl-tRNA Synthetase
(dimer of identical subunits in green and
blue - tRNAs in red)
Representative Class I (left) & Class II (right) structures
Different classes bind to
different “sides” of the tRNA
tRNA
Class I enzyme
Class II enzyme
The Sidedness of tRNA Binding Defines the Reaction Mechanism
tRNA
anticodon loop
Class I enzymes
cradle the tRNA,
gripping the
anticodon loop, and
placing the amino
acid acceptor end of
the tRNA in the
active site (at the top
right in each tRNA).
These all approach
the tRNA similarly
and add the amino
acid to the last 2’OH
group in the tRNA.
The Sidedness of tRNA Binding Defines
the Reaction Mechanism
The Class II enzymes
such as the
phenlyalaninyl tRNA
synthetase approach the
tRNA from the other side,
and add the amino acid to
the 3’ hydroxyl on the last
tRNA base.
http://www.rcsb.org/pdb/molecules/pdb16_2.html
Aminoacyl-tRNA Synthetases
• All have a common 2-domain structure
A catalytic domain
A variable domain
Interacts with the
tRNA 3’OH
Interacts with the
specific bases on the
tRNA that identify
that tRNA
Recognizes and
binds the cognate
amino acid
Aminoacyl-tRNA Synthetases
• Must exhibit high specificity (fidelity)
• This is a two different levels:
1) They must be able to recognize and
bind to the correct tRNA
2) They must be able to recognize and
bind to the correct amino acid (have an
editing function for this one)
Recognition of tRNAs
Some tRNA synthetases
recognize their cognate
tRNA by binding to the
anticodon loop.
These enzymes are not
gentle with tRNA molecules.
For example glutaminyl-tRNA
synthetase firmly grips the
anticodon, spreading the
three bases widely apart for
better recognition.
Recognition of tRNAs
• Recognition of tRNA molecules using the anticodon
is not always possible.
• For example 6 different codons specify Ser, so seryltRNA synthetase must recognize six tRNA molecules
with six different anticodons (isoacceptor tRNAs).
• Therefore, tRNA molecules are also recognized
using bases elsewhere in the molecule.
• Base number 73 in the sequence, seems to play a
major role in many cases, but in other cases it is
completely ignored.
tRNA structure and recognition elements
Recognition of tRNAs
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:
1) one or more bases in anticodon,
2) one or more bases in the acceptor stem,
3) discriminator base 73
Identity elements in tRNAs
tRNAMet
tRNAVal
Identity elements
reside in the anticodon
Alterating the anticodon
of tRNAVal to the Met
anticodon results in
recognition of the mutant
tRNA by methyonyl-tRNA
synthetase
Identity elements in tRNAs
tRNAPhe
The 5 bases that are
identity elements
reside in the anticodon
(3), G20 in the D loop
and A73 near the 3’end
G20 may be especially
important in recognition
of tRNAPhe since it is not
found in any other tRNA
Identity elements in tRNAs
tRNASer
Six codons for Ser,
which are quite different
from one another.
Six “isoacceptor” tRNAs
It makes sense that the
anti-codon loop is not
used to recognize
tRNASer
Identity elements in tRNAs
tRNAAla
Single G3:U70 pair
defines specificity
G:C, A:U or U:G do not
work
Including this base
pair in other tRNAs
allows them to be
recognized by the
alanyl tRNA synthetase
A completely synthetic
“microhelix” can be
aminoacylated provided that
G3:U70 is present
tRNAAla
Single G3:U70 pair
defines specificity
G:C, A:U or U:G do not
work
Including this base
pair in other tRNAs
allows them to be
recognized by the
alanyl tRNA synthetase
High fidelity in amino acid
selection
•Aminoacyl-tRNA synthetases must perform their tasks with
high accuracy, since every mistake will result in a misplaced
amino acid when new proteins are constructed.
•These enzymes make about one mistake in 10,000. For most
amino acids, this level of accuracy is not too difficult to achieve.
•Most of the amino acids are quite different from one another.
•But in a few cases, it is difficult to choose just the right amino
acids and these enzymes must resort to special techniques.
http://www.rcsb.org/pdb/molecules/pdb16_3.html
High fidelity in amino acid
selection
•Isoleucine is a particularly difficult example.
•It is recognized by an Ile-shaped hole in the enzyme, too
small to fit larger amino acids like Met and Phe, and too
hydrophobic to bind anything with polar side chains.
•But, the slightly smaller amino acid Val, different by only a
single methyl group, also fits nicely into this pocket, binding
instead of Ile in about 1 in 150 times.
•This is far too many errors, so corrective steps must be
taken.
http://www.rcsb.org/pdb/molecules/pdb16_3.html
High fidelity
•Isoleucyl-tRNA
synthetase solves this
with an editing site.
•Ile does not fit into this
site, but Val does.
•The mistaken Val is then
cleaved away, leaving the
tRNA ready for a
properly-placed Leu
amino acid.
•This proofreading step
improves the overall error
rate to about 1 in 3,000.
http://www.rcsb.org/pdb/molecules/pdb16_3.html
3’ 5’
CGG
Codon-Anticodon
Interaction
Anticodon loop
3 2 1
1 2 3
5’
GCC
Codon in mRNA
3’
• Predict that every codon should have a corresponding tRNA
(and anticodon)
• BUT IT IS NOT THAT SIMPLE
• There are 61 different codons specifying amino acids
• BUT there are far fewer than 61 different tRNAs
• we can conclude that some tRNAs must bind to > 1 codon
Codon-Anticodon Interaction
For Example:
Yeast tRNAAla
GCU
GCC
GCA
• Recognition of the 3rd codon base is not as “precise” as the
first 2 codon bases
• The genetic code is consistent with this (3rd base
degeneracy)
This led Crick to propose a “Wobble Hypothesis”
-there are canonical base pairs for the 1st and 2nd bases of
the codon, and non-canonical base pairs for the 3rd base
Codon-anticodon interactions
Anticodon
(base #1)
Codon
(base #3)
C
A
G
U
I
G
U
C,U
A,G
U,C,A
• with these rules a minimum of 31 different tRNAs is required
to recognize all 61 codons that encode amino acids
Third-Base Degeneracy
and the Wobble Hypothesis
• Codon-anticodon pairing is the crucial feature of
the "reading of the code"
• But what accounts for "degeneracy": are there 61
different anticodons, or can you get by with fewer
than 61, due to lack of specificity at the third
position?
• Crick's Wobble Hypothesis argues for the second
possibility - the first base of the anticodon (which
matches the 3rd base of the codon) is referred to
as the "wobble position"
The Wobble Hypothesis
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
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
• Preferred codons depend on A:T/G:C content
• 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
Nonsense suppression
• Mutations that produce in-frame TAA, TAG and TGA
result in premature termination of protein synthesis
• Second mutations may appear that suppress the
effect of nonsense mutations: these suppressors are
tRNAs!!!
• In these tRNA mutations, anticodons are altered so
that “stop” signals could insert amino acids
• Suppressor tRNAs originate from minor isoacceptor
tRNAs so that they do not interfere with translation of
highly utilized codons.