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Error Rates of the Replication and Expression of Genetic Information.

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Error Rates of the Replication and Expression of Genetic
Information
By Uwe Englisch,* Dieter Gauss, Wolfgang Freist, Sabine Englisch,
Hans Sternbach, and Friedrich von der Haar
Dedicated to Professor Friedrich Cramer
Biological evolution is based on continual changes in the genomes of cells. If stable organisms are to develop nonetheless in the course of this process, low error rates (high accuracy)
in both the replication of genes and their transcription and translation into proteins have to
be assured. The overall error rates of these processes are measurable and can be estimated:
they result from the error rates of the individual enzyme-catalyzed steps. In many cases, for
example in the discrimination of the amino acids valine and isoleucine during protein biosynthesis, it is not possible to achieve a sufficient accuracy only on the basis of the difkrence of the free energies of binding of the correct and incorrect substrates (the two amino
acids differ only in a CH2 group). The necessary low error rate is maintained by a n additional proofreading step, which is carried out by the enzymes involved after formation of
the enzyme-substrate complex, either during catalysis or before product release. In this step,
an incorrect intermediate o r product is hydrolyzed. The energy input necessary for the synthesis of the incorrect intermediate or product, which is provided by hydrolysis of adenosine or guanosine triphosphate, is the price paid for the low error rates. In this article, the
proofreading mechanisms of several enzymes-mainly those involved in DNA replication
and the aminoacylation of transfer RNA during protein biosynthesis-are discussed.
1. Introduction’**’
The specificity of enzymes has fascinated chemists and
biologists since the early days of bioorganic chemistry.
Emil Fischer’s “lock and key” theory of enzyme-substrate
recognition was the first mechanistic model of this interaction.[’] The rapid development of biochemistry in the last
few decades has resulted in increasingly detailed models of
enzymatic
such as the “induced fit” theory
(fitting of the active site of an enzyme to the substrate) and
the “strain” theory (activation of the substrate by the enzyme). At the same time the principles of enzymatic catalysis have been evaluated using low-molecular-weight organic
The elucidation of the basic molecular mechanisms of heredity and the formation of phenotypes has stimulated the search for enzyme systems responsible for DNA replication and protein bio~ynthesis[’~’~
(see
Fig. 1). Shortly after their discovery it was demonstrated
that these enzymes exhibit greater specificity”. lo] than most
of the known enzymes of such important metabolic pathways as the Calvin, citrate, or urea cycles.
[*] Dr. U. Englisch, Dr. I). Gauss, Dr. W. Freist, Dr. S . Englisch,
[**I
Dr. H. Sternbach
Abteilung Chemie, Max-Planck-lnstitut fur experimentelle Medizin
Hermann-Rein-Strasse 3, D-3400 Gottingen (FRG)
Prof. Dr. F. von der Haar
Geschaftshereich Medizin- und Labortechnik, 8 . Braun Melsungen
AG
Postfach I 1 10. D-3508 Melsungen (FRG)
Recent reviews (added in proof, Oct. 7, 1985): A. R. Fersht, Adu. Exp.
Mrd. 17Y (1984) 525; F. Grosse, G . Kraus, J. W. Knill-Jones, A. R.
Fersht, ihid. 17Y (1984) 535; H. Echols, Bioessayr I(1984) 148; M. Yarus, R. C . Thompson in J. Beckwith, J. Davies, J. Gallant (Eds.): Gene
Funcrian in Prokaryxrs. Cold Spring Harbor 1983, p. 23; L. BrakierGingras, P. Phoenix, Can. J . Biochem. Cell Bid. 62 (1984) 231.
Angew. Chem I n t . Ed. Enql. 24 (1985) 1015-1025
In 1963 Loftfield investigated the accuracy of ovalbumin
biosynthesis in hen oviduct tissue1”I and showed that the
misincorporation of valine in place of isoleucine occurred
with a frequency of = 1 :3000. This overall error rate includes all possible errors in protein biosynthesis, from the
transcription of DNA through the esterification of amino
acids to transfer RNAs u p to peptide bond formation at
the ribosome.
The error rates of DNA replication in prokaryotic and
eukaryotic cells, o n the other hand, are orders of magnitude lower, as indicated by the rates of spontaneous mutation.”’, 131 They occur in the range of one mutation per
= 10’ to 10” incorporated nucleotides, which reflects the
extreme accuracy with which the genetic code is conserved. However, this error rate does not only include the
errors of the D N A polymerase, but also all other changes
of the DNA s e q ~ e n c e [ ’ ~that
- ’ ~ are
~ not corrected by repair
enzymes before replication starts.[”I Such changes can be
caused, for example, by UV radiation (dimerization of
neighboring thymidines to form a cyclobutane derivative)
or by mutagenic compounds (alkylation, deamination of
nucleosides.
The extremely low error rate of DNA replication, however, need not be achieved in gene expression, since in replication each nucleic acid strand is replicated only once,
whereas in gene expression one gene is transcribed to give
many copies of messenger RNA, which, in turn, serve as
templates for the synthesis of many protein molecules.
Both the messenger RNAs and the proteins are degraded
after a short lifetime. Moreover, proteins with a wrong amino acid sequence often have a different tertiary structure
which favors fast proteolytic cleavage.[’”
0 VCH Verlagsgesell.whafi mhH. 0-6940 Wernheim. 1985 0570-0833/85/1212-/01S S 02.50/0
1015
G-T-T-4-T-IJEA- A-T-A-AiG-I-SC-A-A-
DNA polvmerase:
dGTP/dATP/dCTP/TTP
-9-1-T-G-T-p-T-A-A-C-A-A-
GTP/ATP/CTP/UTP
-
lie - - \ l a l - - j j l
1
.1
I
-
-
DNA + pyrophosphate
RNA + pyrophosphate
Translation of mRNA t o protein on t h e ribosome
Aminoacyl-tRNA synthetase:
amino acids + ATP + tRNA
aminoacyl-tRNA + AMP + pyrophosphate
-
-
t idase e t c. (a f f er codon-ant icodon r ecog nit ionl:
aminoacyl-tRNAs + nGTP
protein + tRNAs + nGDP
Fig. I . Scheme of replication, transcription, and translation; some key enzymes and their reactions with energy-rich substrates are given with the reaction steps.
The expression of the genetic information is a multistep
system optimized for economy and efficiency. Accordingly, the error rates of the single steps are always expected to
be slightly lower than the overall error rate, which is obtained (to a first approximation) by the addition of the single error rates. However, if the accuracy of any one step is
improved by a proofreading mechanism, the resulting error rate (again to a first approximation) is the product of
the initial discrimination of the substrates and a proofreading factor, which is the inverse ratio of the number of errors before and after the proofreading.
nism is responsible for an increased rate of mutation in a
rapidly changing environment.[241
A low accuracy in protein biosynthesis plays a major
role in one of the theories of biological aging, the “error
catastrophe theory” of OrgeZ.‘2s1Since all reaction steps of
the protein biosynthesis are catalyzed by enzymes, errors
in the synthesis of these enzymes lead to the biosynthesis
of increasingly defective proteins, resulting finally in cell
death. The error catastrophe is definitely not the only
source of cell aging, but a decrease in enzyme activities
and specificities might play an important role in this process.[26-311
2. Relevance of Error Rates in the Replication and
Expression of Genetic Information
High error rates during DNA replication and protein
biosynthesis would have two important consequences.
First, a high (but not lethal) exchange rate for single nucleotides during DNA synthesis would generate many different mutants. This would enable organisms to adapt faster to changing environmental conditions, assuming a sufficient number of cell divisions occur. Second, the stability
of the genome wouId be impaired and usefuI mutations
would not be conserved. In gene expression high error
rates would lead to large amounts of heterogeneous, nonspecific, functionally impaired, or even nonfunctional proteins. The consequences would range from metabolic disturbances to possible breakdown of the cell metabolism.
The accuracy of DNA replication and protein biosynthesis are coupled to each other. On the one hand, errorprone DNA replication results in a direct change of the
genotype, leading to defective proteins, even if the protein
biosynthesis is free of errors. On the other hand, errorprone protein biosynthesis results in DNA polymerases
and other proteins that interact with DNA with low speciticity. The necessity of maintaining intact replication units
and simultaneously permitting a certain level of mutations
led in the course of evolution to an optimized range of mutation rates. Slow and fast mutating species of one organism are known‘20-231
in which the mutation rate is geneti:ally determined or in which a specific regulation mechaSO16
The severe effect of one point mutation in a large genome is exemplified in sickle cell anemia:[321
persons carrying homozygotic chromosomes have a shorter lifetime due
to a change in the shape of their erythrocytes. This phenomenon is the result of an amino acid exchange in position 6 of the @chain of hemoglobin, where a valine is inserted in place of a glutamic acid. The sequence CTC in
the gene, which is transcribed into GAG in mRNA, codes
for glutamic acid.[33’Since valine is coded for by GUG in
mRNA, the corresponding sequence in the gene is CAC,
indicating a T+A exchange.
Recent studies on tumor celI Iines show a connection between point mutations and tumor induction: in cell lines of
human bladder carcinoma, one uncorrected point mutation changes a silent proto-oncogene into an active oncogene. This one mismatch produces a glycine/valine exchange in the expression p r o d u ~ t , [ ~which
~ - ~ ~isI a key factor for the transformation of the cell.
3. Error Rates of DNA Replication
The synthesis of a daughter strand of the DNA double
helix is catalyzed by DNA polymerase with the assistance
of many other proteins. The polymerase uses the deoxynucleotides dATP, dCTP, dGTP, and TTP as substrates,
while the mother strand serves as template (Fig. 1). The
enzyme does not recognize single deoxynucleotides, only
base pairs formed with the pyrimidine and purine residues
of the template.[38JThe accuracy of this selection process is
Angew. Chem. h i . Ed. Engl. 24 (1985) 1015-1025
not perfect, as the shapes and properties of the correct
base pairs can be mimicked. Watson and CrickL3’]suggested that amino/imino and keto/enol tautomerism of
the nucleobases can reduce the specificity of DNA polymerases. The imino form of cytosine can establish the
same hydrogen bonding pattern as thymine and the enol
form of thymine that of cytosine, resulting in A :C (imino)
and G :T(enol) base pairs. Additionally, the interatomic
distances in these mismatch base pairs are the same as in
the correct A :T and G :C base pairs (Fig. 2).
thymine
adenine
H
cytosine
guanine
H
thymine, enol form
big. 2. Structures 01 thymine.adenine and cytosine.guanine base pairs in
comparison with the imino form of cytosine and the en01 form of thymine,
which are suitable for mispairing. rib = ribosyl.
This mispairing occurs with a frequency of = 1 : lo4 to
1 :105.[40*4’1
This rate is by 3 to 6 orders of magnitude
higher than the frequency of in-vivo mutation of E. coli
(= 1 : 10’ to 1 : 1010).1421
The mutation rate in eukaryotic
cells is thought to be even lower by a factor of about 100.
The slower rate of replication (30 nucleotides/s versus
1000 nucleotides/s in E. coli) may be an indication that
eukaryotic cells use more sophisticated proofreading steps
before a nucleotide is finally incorporated into the
daughter ~ t r a n d . ’ ~ ~ . ~ ]
the enzyme, which seems to control the exonuclease activity, is responsible for the observed high rates of mutation.148.491
The quantitative measurement of error rates in DNA replication-using as templates either synthetic polynucleotides in in-vitro biochemical assays[451or DNA in in-vivo
molecular genetics assays[50s511-hasshown that the error
rate is reduced by a proofreading system to one mistake
per = lo6 to lo9 polymerized n u c l e ~ t i d e s . [ ~The
’ - ~ ~proof~
reading system of prokaryotic DNA polymerases therefore
decreases the error rate by a factor of = l o 2 to lo4 compared with the rate of mispairing but this is still not sufficient to explain the observed rates of spontaneous mutations. A further reduction is achieved by the action of a
second system, the post-replicative proofreading.
Until recently, it was thought that error correction could
only occur before the incorporation of the next nucleotide
because the proofreading enzyme cannot distinguish afterwards which nucleotide in the mismatch base pair was the
original correct one. However, it has been shown in the
meantime that specific methylation of the DNA strand,
which occurs in all cell systems examined so far,[”] provides a recognition signal to differentiate between mother
and daughter strands. The methylation of adenine (N6
atom) in the sequences G-A-T-C of E. coli (Fig. 3 ) lags be-
H-0-H
Hd 6H
Fig. 3. Enzymatic methylation of DNA; this reaction allows the template
DNA strand to be discriminated from the newly synthesized strand.
3.1. Mechanism of Proofreading in DNA Replication
To maintain a low error rate of DNA replication, prokaryotic DNA polymerases have an exonuclease activity in
addition to their polymerizing activity. In the case of a recognized mismatch, excision of the incorrect nucleotide
takes place. Only after this proofreading step and, if necessary, incorporation of the correct nucleotide does replication continue.[44-461The connection between polymerase
and exonuclease activities was demonstrated for the first
time when fast- and slow-mutating species of bacteriophage T4 (“mutator” and “antimutator” strains) were examined.[471The exonuclease activity of DNA polymerase
isolated from “mutator” phages is much lower than its polymerase activity. Therefore, the proofreading activity of
this enzyme is low in comparison with that of the wild-type
enzyme. Exactly the opposite phenomenon was observed
with the “antimutator” phages. In further studies with
DNA polymerase 111 from a mutator E. coli strain it could
be shown that a mutation in the gene of the &-subunit of
Angew. Chem. Int. Ed. Engl. 24 (198s) 1015-1025
hind
such that newly synthesized strands are
transiently unmethylated or undermethylated. The mismatch repair system moves along the undermodified
daughter strand, removes the mismatches, and replaces
them with the correct nu~leotides.[’~-~’~
This mismatch repair system reduces the error rate of replication by a factor
of = 100 to 10000, resulting in an overall error rate of one
mistake in = lo9 to 10” polymerized nucleotides.
4. Error Rates of Transcription
The transcription of the genetic information from DNA
into mRNA by DNA-dependent RNA polymerase does
not appear to involve a proofreading
The observed in-vitro error frequencies with synthetic polynucleotides are in the range of one mistake per = lo3 to 10’
polymerized ribonucleotides ;[621 for example, in an in-vivo
E. colz system the transcriptional error of a nonsense muta1017
tion is one mistake per 1 O4 polymerized r i b o n u c I e o t i d e ~ . [ ~ ~ ~ details of this process are still unknown, energy might be
This error frequency is the same as that predicted from the
necessary for providing the correct stereochemical orientaintrinsic tautomerism of the heterocyclic bases, and thus
tion of the macromolecules, and for assuring the high rate
no evidence for a proofreading mechanism is detectable.
of elongation of the peptide (i= ten aminoacyl residues per
second).
5. Error Rates of Aminoacylation of Transfer RNA
5.1. Selection of Transfer RNA
The main task of the enzymes involved in replication
and transcription is to assure the correct base-pairing of
nucleotides. In protein biosynthesis, on the other hand,
three nucleotides (triplet or codon) of the messenger R N A
(mRNA) have to be correlated to one amino acid (cf. Fig.
1). The links between these two systems are the transfer
RNAs ( ~ R N A S ) , ~which
~ ~ - ~consist
~]
of 50 to 100 ribonucleotides. In Figure 4 (bottom left), the schematic structure
of phenylalanine tRNA is shown. The binding of the
tRNA to the mRNA occurs by base-pairing of the three
nucleotides of the anticodon with the codon of the
mRNA; the corresponding amino acid is esterified to the
3'4erminal adenosine of the tRNA (cf. Fig. 1 and Fig. 4).
The aminoacylation of each tRNA species is catalyzed by
a specific aminoacyl-tRNA s y n t h e t a ~ e . [ ~ ~In- ~this
* ~ reaction, the following two selection problems arise for each of
the twenty enzymes: the accurate selection of the correct
tRNA from a population of at least 20-but usually ca.
70-species, and the accurate selection of the correct amino acid from 20 po~sibilities.['~]
The aminoacylation takes place via two successive steps
known as activation and transfer (Fig. 4). The intermediate
after activation of the amino acid by ATP, the aminoacyl
adenylate, is a mixed anhydride. Thermodynamic considerations show that the free energy of hydrolysis of the a$phosphoric acid anhydride bond of ATP is largely conserved in the free energy of hydrolysis of the aminoacyltRNA.l7'] This large amount of en erg^['^.^'] may be necessary for the subsequent transformation of the ester linkage
into a peptide linkage at the ribosome. Although the exact
amino acid
AA
+
adenosine triphosphate
+
ATP
.......
..............
"
...
"
':a
20
R-C-C-0-P-0
L
26
'AH3
F1
@b
38
31
antlcodon
-
HO
+
+
OH
aminoacyl adenylate
AA-AMP
+ pyrophosphate
PP
+
................
' Adei
de+
-
aminoacyl a d e n y h t e
AA-AMP
...
-
Each aminoacyl-tRNA synthetase has to select its cognate tRNA, carrying the amino-acid-specific anticodon.
However, the anticodon does not seem to be the recognition site-at least not the only one-of the tRNA for the
synthetase. Although many studies have been carried out,
e.g., aminoacylations of chemically or enzymatically modified tRNAs, the addition of cross-linking reagents to aminoacylation reactions, the misaminoacylation of tRNAs, or
the comparison of the ca. 1000 known tRNA sequences,[761
no specific recognition site on the tRNA (Fig. 4) has been
Th.IS clearly demonunambigously identified.165*67.6x,73.771
strates the complexity of the selection process, which probably changes in terms of both the recognition site and the
selection mechanism from tRNA to tRNA and from organism to organism. The enzyme (consisting of 500 to 1000
amino acids) and the tRNA (50 to 100 nucleotides) offer a
large number of possibilities for recognition. Taking into
consideration the informational content of an anticodon or
codon, a recognition sequence of six steps is reasonable if,
for each of the three base pairs, two questions must be
posed: Is the opposite base a purine or a pyrimidine? In
the former case, is it A or G, and in the latter, U or C ?
Accordingly, six questions arise for a sequence of three
base pairs. The results of biochemical e ~ p e r i m e n t s [ ~ ~ - ~ ~ ]
and theoretical s t u d i e ~suggest
~ ~ ' ~ that
~ ~ a~ similar number
of steps are involved in the recognition process. Probably,
step by step, the cognate tRNA docks more and more
tightly to the e n ~ y m e . [ ~ An
~ - *important
~~
role-at least in
some systems-is played by the terminal adenosine of the
t
00-pI
QO
...............i
IHO OH
transfer-RNA
tRNA
aminoacyl-transfer-RNA
AA-tRNA
-OVe
HO
OH
+ adenosine monophosphate
+
AMP
Fig. 4. The coupling of tRNAs with amino acids (aminoacylation), catalyzed by aminoacyl-tRNA SynthetdseS, is a two-step process: the formation of an aminoacyl
adenylate from an amino acid is called activation (upper reaction); aminoacyl-tRNA formation is called transfer (lower reaction). Bottom left: sugar-phosphate
backbone of yeast tRNAPhC(76 nucleotides).
1018
Angew. Chem. Int. Ed. Engl. 24 (1985) 1015-1025
tRNA and its 2‘- or 3‘-hydroxyl g r o ~ p s , [ ~to~which
, ~ ~ ] the
cases, this
aminoacyl residue is t r a n ~ f e r r e d . [ ~ ~In- ~most
”
where k,,, and K , are the turnover numbers and the Mitransfer specifically involves one of these hydroxyl groups ;
chaelis constants, respectively, of the substrates and AAG
the other one-at least in several cases (see Section 5.2)is the difference between their free energies of binding in
is important for the error rate of the aminoacylation. In
the transition state. According to calculations based on the
most experiments, an error rate of one mistake per = lo4 to
London theory of electronic dispersion forces and on
lo5 tRNA-enzyme recognitions is observed, which is in
measurement of energy differences in binding of chemithe range of the other steps of gene expression and therecally related compounds to antibodies,
defore does not seem to require a proofreading step. As early
monstrated in the 1950s that the free energy for the hydroas 1972/ 1973, however, it was demonstrated that phenylalphobic interaction of a methylene group with a molecule is
anyl-tRNA synthetase from E. coli could hydrolyze misamabout 4 kJ/mol. Using this result, he calculated, years beinoacylated Ile-tRNAPhe,the product of a misaminoacylation reaction using isoleucyl-tRNA ~ y n t h e t a s e ; ~this
~ ~ , ~ ~ ] fore the discovery of the molecular mechanism of protein
biosynthesis, that the error rate for the incorporation of
hydrolysis was called “verification.” Detailed examination
glycine instead of alanine, or valine instead of isoleucine,
of the recognition process in the system isoleucyl-tRNA
Although another calculation in the
must be about 1 :
synthetase + tRNA”= + tRNAValfrom yeast (the close re1970s gave a lower error rate,”” these numbers are very far
lationship of the two tRNAs is quantified by the number
from the error rates necessary in protein biosynthesis.
and kind of the ribonucleotides) recently showed that the
initial discrimination of the tRNA is only = 1 : 10 and that
an additional proofreading step is necessary to decrease
ttRNA1le
I l e + ATP t E l l e \
E1le.Ile-AMP t PP
the error to -- 1 : 100.[901In vivo the error is then diminished
to = 1 : lo4 to 10’ by the presence of the tRNAVa’-complexE l l e t AMP
ing valyl-tRNA synthetase. Nothing is known yet about
the mechanism of this proofreading.
~
t(Ile-tRNAIle1
5.2. Selection of the Amino Acid
Val
In comparison to tRNAs, amino acids are very small cell
constituents. In addition to their small size, some of them,
e.g., glycine and alanine, phenylalanine and tyrosine, or
isoleucine and valine (Fig. 5), are very similar. Therefore, it
may be especially difficult for the enzyme to differentiate
very accurately between the closely related amino acids.
@
coo@
I
H3N-Y-H
@
cocp
I
H3N-C-H
H
CH3
@
coo@
I
coo@
0
H3N-C-H
H-C-OH
H-C-OH
I
I
H
GlY
@
Ala
coo@
I
H,N-c-H
I
/ CH
\
H3C
coo0
I
H,~--c-H
CH3
1
H3N-C-H
CH3
Ser
@
coo@
I
H3N-C-H
I
H&-C-HI
7H2
CH3
I
Thr
@
coo@
I
H3N- C-H
I
6 6
OH
Val
I le
Phe
ATP
+
E’’~-+
ttRNArle
EI’~.V~I-AMP
t PP
~
E1le
t
AMP + ( V a l
*
tRNA1lel
Fig. 6. Aminoacylation of tRNA”‘ with isoleucine by isoleucyl-IRNA hynthetase.(E”‘, above) and misactivation of valine by the same enzyme (followed
by hydrolysis to valine and tRNA”‘)
The difficulties predicted by Pauling for the discrimination between isoleucine and valine by the isoleucyl-tRNA
synthetase of E. coli were confirmed experimentally some
years later: valine is actually misactivated by the enzyme to
give enzyme-bound valyl a d e r ~ y l a t e [ (Fig.
~ ~ , ~6,~ ]cf. Fig. 4,
top). Berg et a1.[9.95,97,981
showed that, despite this activation, n o Val-tRNA”’ is obtained in the presence of tRNA”‘
(Fig. 6). In 1972, however, Val-tRNA”’ was demonstrated
to be the probable intermediate.[991The mechanism of this
reaction remained unclear for some time. The product is
apparently proofread by the enzyme, and a misactivated
amino acid is eliminated by hydrolysis and not released as
aminoacyl-tRNA from the enzyme. The cost of this proofreading is the hydrolysis of one ATP molecule to AMP per
misactivation (Fig. 6).
TYr
Fig. 5. Satisfactory discrimination of some amino acids by aminoacyl-tRNA
synthetases requires proofreading steps. Structures of some related amino
acids: glycine a n d alanine, serine and threonine, valine and isoleucine, phenylalanine and tyrosine.
The decisive parameter determining the ability of an enzyme to discriminate between two substrates (A and B) is
the difference in the free energy of binding of the two substrates in the transition state. Under the assumption that
the reaction steps involved are energetically the same for
both substrates, this parameter can be calculated from relatively easily measurable kinetic parameter^:".^"
Angew. Chem. lnr. Ed. Engl. 24 (1985) 1015-1025
t
5.2.1.
Proofreading Mechanism of Aminoacylation
Numerous experimental results and theoretical arguments on the mechanism of proofreading by aminoacyltRNA synthetases have led to different models for this
phenomenon. Each of these, however, was only developed
for one or a few of the aminoacyl-tRNA synthetases and,
therefore, should not be regarded as general or valid in all
proposed a correction
cases. In 1974, Hopfield et al.[’oo~’o’l
mechanism, called “kinetic proofreading.” The main principle of this mechanism is that the discrimination between
two competing substrates, according to Michaelis-Menten
1019
kinetics, can be repeated on the level of the intermediates,
resulting in an error rate that is the product of the error
rates of the two steps. This argument is only valid if there
is a n irreversible step between the Michaelis-Menten complex and the intermediate (Fig. 7, step 2) and if this inter-
E
t
1
2 - w
e e E . e jE . e
I
3
>-
E t p
+ e'
3 ' , E
1
2
3
+ATP
E
t
A A e E . AA
--+E.AA-AMP
-pp
I
J/
ttRNA
E + AA-AMP
AA-tRNA t AMP t E
3'
+
AA
t
AMP
Fig. 7. A reaction scheme suitable for "kinetic proofreading" (above, taken
from Hopfield) and aminoacylation according to this scheme (below, E=aminoacyl-tRNA synthetase).
mediate can in principal be processed in two different
ways (Fig. 7, step 3 or 3'). In the aminoacylation reaction,
the hydrolysis of ATP (and the subsequent hydrolysis of
pyrophosphate) is regarded as an irreversible step (Fig. 7,
step 2), and the enzyme-aminoacyl adenylate complex can
either react to give product (Fig. 7, step 3) or dissociate
from the complex and undergo hydrolysis in solution (Fig.
7, step 3'). This model is very convincing and is supported
by the experimental fact that significantly increased ATP
consumption is observed in aminoacylation of tRNAs with
noncognate amino acids. For instance, 270 molecules of
ATP are hydrolyzed before one molecule of Val-tRNA"'
(E. coli) is formed, whereas the formation of the cognate
Ile-tRNA"' only requires 1.5 ATP molecules.[lOO~
102-1041 So
far, the most persuasive experimental support for such a
model is the discrimination between tyrosine and phenylalanine by phenylalanyl-tRNA ~ y n t h e t a s e . ~This
' ~ ~ ] mechanism of selection is probably also of importance for other
amino acids (cf. Section 5.2.2). The obvious weakness of
the model is that it only deals with a proofreading mechanism on the level of the aminoacyl adenylate and does not
explain what happens on the level of enzyme-bound transient misaminoacylated tRNAs, which have been detected
by several group^.^^^,'^^, 106-1081
Two other proofreading models d o take into consideration these species. The double sieve model of Fersht et
al~[109-llZjIS
. a two-step mechanical model, in which the active site acts twice as a sieve for the selection of the correct
amino acid. The first sieve, the binding pocket, which perfectly accomodates the amino acid, blocks the binding of
more voluminous amino acids by steric repulsion. However, smaller or isosteric amino acids are bound (albeit with
lower affinity) and can react to give the aminoacyl adenylate. The second sieve acts in the following way: after
transfer of the aminoacyl residue to the accepting hydroxyl
group of the terminal adenosine of tRNA (cf. Section 5.1
and Fig. 4), isomerization to the nonaccepting hydroxyl
group (which is close to a hydrolytic site of the enzyme)
occurs if the aminoacyl residue is smaller than the correct
1020
one. The cognate aminoacyl residue cannot reach this site
for stereochemical reasons and remains bound to the
tRNA. The double sieve model is the result of experim e n t ~in~which
~ ~ fast
~ kinetics
. ~ ~ were
~ ~ used to detect the
intermediates. Proofreading could be found with methionyl-,[l'ol valyl-,[109.111~1151
and isoleucyl-tRNA synthetases[Io4.Io7] from E. coli and Bacillus stearothermophilus.
The authors concluded that, in the case of isoleucine," "I
t h r e ~ n i n e , [ ' ~and
~ ] a-aminobutyric acid,[1151the proofreading mechanism acts mainly after transfer of the amicoacyl
residue to the tRNA, whereas valyl adenylate is hydrolyzed before the transfer step. The model could not explain, however, the activation, transfer to tRNA""', and final hydrolysis of the valine-isosteric threonine by the valyl-tRNA synthetase without further assumptions. The discrimination of more voluminous amino acids in the first
sieve has been cast in doubt by the observation of a misactivation of cysteine by alanyl-tRNA synthetase.['l6I
Using chemically modified tRNAs, in which either the
2'- o r 3'-hydroxyl group was missing or replaced by a n amvon der Haar, et al.[119-1211
were
ino g r o ~ p , [ " ~ ~ Cramer,
"*~
able to show that, at least in yeast, both hydroxyl groups
act together in the proofreading.[122,1231
Besides the esterification of isoleucine with tRNA1Ie to He-tRNA""-C-C-A
(Fig. 8a), isoleucyl-tRNA synthetase also catalyzes the formation of misaminoacylated Val-tRNA1'"-C-C-3'dA, which
is likewise released from the enzyme. In contrast, ValtRNA1Ie-C-C-Ais hydrolyzed by the enzyme before release
can occur.[103~1241
The model of chemical proofreading explains the hydrolysis of Val-tRNA""-C-C-A by the assumption that the nonaccepting 3'-hydroxyl group and a
water molecule-which replaces the missing methylene
group-together with a nucleophilic group of the enzyme
are responsible for the hydrolysis of Val-tRNA"'-C-C-A
(Fig. 8b). Although the fact that one ester is hydrolyzed
and the other is not can be explained quite simply for this
specific case, this model cannot be easily generalized to
other systems. Investigations of the valine system (Fig. 8c)
showed that the hydrolysis of a mistransferred threonyl residue by valyl-tRNA synthetase (Fig. 8d) is not directly
mediated by the 3'-hydroxyl group of the terminal adenosine of the tRNA.[106,1091
A possible explanation for this
phenomenon would be the transient lactonization of
threonine (Fig. 8d), but since the four-membered lactone
product is unstable, no direct chemical proof is possible.
Although a stable five-membered lactone could be formed
from y-hydroxyvaline (which is roughly the same size as
isoleucine) by isoleucyl-tRNA synthetase, the enzyme does
not accept this analogue as a
However,
lactones are formed in two cases where the substrate is too
voluminous (in the sense of the double sieve) by one methylene group, namely from y-hydroxyvaline by valyltRNA synthetase (Fig. 8e)"25,1261and from y-hydroxyisoleucine by isoleucyl-tRNA ~ y n t h e t a s e . [ ~Both
~ ~ " ~reac~~
tions are enzyme-catalyzed in yeast as well as in E. coli. In
addition, homocysteine, a precursor of methionine, is released as a thiolactone by methionyl-tRNA synthetase (E.
C O I Z ) [ ~(Fig.
~ ~ ~ 8g). These examples show that an intramolecular nucleophilic attack of the hydroxyl group of the amino acid on the carboxyl group is involved in the ester hydrolysis.
Angew. Chem. Int. Ed. Engl. 24 (198s) 1015-1025
CH3
S
/ \
HzC
C=O
+
I
1
0
HzC-CH-NH3
Fig. 8. Schematic representation of aminoacyl-tRNAs in the active sites of aminoacyl-tRNA synthetases: isoleucyl-tRNA synthetase and tRNA"' with the amino
acids isoleucine (a) and valine (b); valyl-tRNA synthetase with the amino acids valine (c), threonine (d), and y-hydroxyvaline (e); methionyl-tRNA synthetase and
tRNAMe'with the amino acids methionine (0 and homocysteine (g).
Many other suggestions regarding the mechanism of
proofreading are found in the literature, in which specific
points of the reaction pathway,['291the stability of intermediates and product^,^'^^^ the interaction of the subunits of
aminoacyl-tRNA syntheta~es,['~'I
and the interaction of
two tRNA molecules simultaneously bound to the enzyme
are
or high-energy conformational states of
the enzyme are p o ~ t u l a t e d [ 'as
~ ~possible
l
ways to differentiate between correct and incorrect substrates. However,
there is no conclusive experimental support for these
ideas.
All measurements and calculations have in common a
final error rate of about one mistake in lo5 aminoacylations, a frequency which is ten times smaller than the overall error rate in protein biosynthesis. This is achieved by
multiplication of error rates at three points: the initial discrimination of the amino acids by the synthetase, the
proofreading of the aminoacyl adenylate (pre-transfer correction), and the proofreading of the aminoacyl-tRNA
(post-transfer correction). Not every enzyme necessarily
uses both pre- and post-transfer proofreading to select its
cognate substrate. Recent investigations have shown, however, that isoleucyl-tRNA synthetase from yeast uses both
steps in the discrimination between isoleucine and valine.
The overall error rate, after the initial discrimination and
both proofreading processes, is about 1 :40000
(10 x 20 x 200).['021
Probably, the error rates in protein biosynthesis are in
the same range for all organisms, although detailed data
are still lacking. What is known, on the other hand, is that
Angew. Chem. Inr. Ed. Engl. 24 (1985) 1015-1025
the mode and extent of the proofreading step has undergone change during evolution: phenylalanyl-, isoleucyl-,
valyl-, and leucyl-tRNA synthetases from different organisms show various combinations, ranging from very accurate initial discrimination and less-expressed proofreading
up to sluggish initial discrimination and very accurate
proofreading, which itself can be pre- or post-transfer only
or a combination of b ~ t h . [ ' ~ ~ Th
, 'e~different
~ , ~ ~proof~ - ~ ~ ~ ~
reading capacities of synthetases from E. coli. fungi, and
mammals with respect to amino acid analogues is one possible starting point for the directed synthesis of therapeutic
agents with protein biosynthesis as the specific target.['371
5.2.2. Aminoaqlation without Proofreading
Besides those aminoacyl-tRNA synthetases that have
proofreading mechanisms (which include, according to
Fersht,I"*I those specific for alanine, valine, threonine, isoleucine, methionine, and, perhaps, phenylalanine and leucine), another class of synthetases exists for which no
proofreading steps have been detected up till now. The
reason for the lack of such proofreading systems is a very
high initial discrimination of the amino acids, which already reduces the error rate below the overall error frequency of protein biosynthesis (= 1 :3000). Examples of
this type of synthetase are the hi~tidyl-,"~~]
tryptophanY I - , [ ' ~ ~ I cysteinyl-,"'31 l y ~ y l - , ~and
' ~ ~tyrosyl-tRNA
]
synthet a s e ~ . [ ' ~The
' ~ observed error rates are in the range of
zz 1 : lo4 to 1 : lo5 for the incorporation of tryptophan and
1021
proline instead of histidine by the histidyl-tRNA synthetase, or of phenylalanine instead of tyrosine by the tyrosyltRNA synthetase. The error rate found for the activation of
serine or alanine by the cysteinyl-tRNA synthetase was
even lower, in the range of = 1 : 108.1”31
In the course of evolution, structures that differ sharply
from those of other amino acids may have been selected
for the amino acids that are directly involved in catalytic
processes, e.g., histidine, tryptophan, or cysteine. The incorporation of the wrong amino acid, leading to an inactive enzyme, would thereby be avoided. Nevertheless, the
above-mentioned amino acids are activated to a small extent by other synthetase~.[’~’I
In the course of evolution,
therefore, the optimal balance of energy cost versus accuracy has been established for every amino acid in every
protein.
6. Error Rates of Peptide Formation at the
Ribosome
After release from the aminoacyl-tRNA synthetase, the
aminoacyl-tRNA forms a ternary complex with the elongation factor Tu and G T P and diffuses to the ribosome.
When the correct anticodon is present, the tRNA binds to
the vacant aminoacyl-tRNA site with release of EF-Tu and
hydrolysis of G T P to GDP. The peptidyl-tRNA, which carries the growing protein fragment, occupies the adjacent
peptidyl-tRNA site. It transfers the polymer by peptide
bond formation to the aminoacyl-tRNA (cf. Fig. 1). In the
next step, called translocation, the deacylated tRNA leaves
the peptidyl-tRNA site and the new peptidyl-tRNA binds
to this site with movement of the mRNA by one triplet.
After this reaction sequence, a new elongation cycle can
start. Many proteins are involved in the ribosomal protein
synthesis, which also requires energy provided by the hydrolysis of GTP. It is not fully known what fraction of the
energy input is used for the peptide bond formation, the
codon-anticodon binding, the regeneration of the helper
proteins to active conformations, or for the other steps inVO~Ved.~~42-1451
In the course of protein synthesis errors can occur. First,
the movement of the mRNA on the ribosome by two or
Ribosome
d Ribosome ---+
.mRNA
t
.mRNA
.Tu.GTP
.AA1- t R N A
Ribosome----+
Ribosome&
.mRNA
-mRNA
.AA1- t R N A
.AA1-tRNA
I
Tu.GTP
.Pep-tRNA
four nucleotides instead of the usual three causes a shift in
the reading frame, resulting in a completely different translation product. Second, the binding of a noncognate aminoacyl-tRNA to the mRNA leads to the incorporation of
an incorrect amino acid in the translation product.
The frequency of a frameshift mutation is of the order of
= 1 : lo4 to 1 : lo5, a value that is in the range of the overall
error rate and does not seem to require a proofreading
Step,l14h,1471
The binding of tRNAs that carry an anticodon with one
mismatch with respect to the codon of the mRNA, e.g.,
binding of Leu-tRNALe” to a mRNA that codes for PhetRNAPhe,however, occurs with a frequency of 1 : 10 to
1 : 100.”481This unspecific binding is equivalent to the error
rate in the formation of a complex of two tRNAs by basepairing of their anticodons in ribosome-free in-vitro experiment~.”~
Two
~ ] proofreading models, the “time-delay’’
model of N i n i ~ l ’ ~lso1
’ . and the “kinetic proofreading”
model of HopJieldl’ov,
‘ “ I 1 propose dynamic processes for
the rejection of incorrect aminoacyl-tRNAs after complexation to the ribosome and the mRNA. The key step in the
first model is a conformational change of the tRNA, which
occurs as a result of the codon-anticodon interaction; in
the second model, the energy provided by GTP hydrolysis
plays the major role. The experimentally proven increase
in G T P consumption upon binding of noncomplementary
tRNAs, which is regarded as an indication for the abortion
and restart of the whole process with irreversible loss of
the energy input, favors the kinetic proofreading model,
but does not exclude the “time-delay mechanism” (Fig.
9).r15’.’ 5 2 1
In addition to these models, a third correction mechanism has been discussed, which takes place after the translocation step on the level of the peptidyl-tRNA. According
to this model, incorrect peptidyl-tRNA dissociates quickly
from the ribosome and is hydrolyzed by peptidyl hydrolase,ll 53-1551 Thi s type of correction, however, would result
in a considerable energy loss, determined by the length of
the fragment already synthesized: e.g., the hydrolysis of a
protein containing 250 aminoacyl residues entails the loss
of roughly 1000 GTPs (Fig. 9).
From the G T P hydrolysis data obtained by 77zompson et
al.1’48,1511
and by Kurlund et aI.,[ls6]a reduction of error
-
Ribosome-
Ribosome
-mRNA
.Pep-AA’-tRNA
.mRNA
.Pep-AA 1- t R N A
I
(b)
(a)
. AA1-tRNA
.AA2-tRNA
\1
\1
Ribosome
Ribosome
.mRNA
.mRNA
+
A A l - tRNA
t
P e p - A A l - t RNR
L
P e p t i d + tRNA
Fig. 9. The individual steps of prokaryotic ribosomal peptide synthesis including proofrcading and correction steps before (a) and after (b) peptide bond formation
(mRNA = messenger RNA, T u -GTP=elongation factor.GTP complex, AA-tRNA = aminoacyl-tRNA, Pep-tRNA= peptidyl-tRNA).
1022
Angew. Chem. I R ~ Ed.
. Engl. 24 (1985) 1015-1025
1: lo1*
I
Replication
(prokaryot. )
overall
1 : l o l o to
w i t h o u t p o s t - r e p l i c a t i v e p r o o f r e a d i n g 1 : lo7 to lo8
w i t h o u t p r o o f r e a d i g b y exonuclease 1 : lo4 t o lo5
1:108
1:104
1
I
Transcription
1 : lo4 to
lo5
(no p r o o f r e a d i n g ]
P r o t e i n b i o s y n t h e s i s l o v e r a l l ) 1 : lo3 t o 10'
including T r a n s c r i p t i o n 1 : 10' t o los
1 : lo4 t o
Selection o f amino acid
1 : lo4 to
Selection o f t R N A
3
Codon-anticodon r e c o g n i t i o n 1 : 10 t o
lo5, without proofreading 1 : lo1 t o lo5
l o 5 , without proofreading 1 : 10' to l o 5
1
5
10 , without proofreadlng 1 : 10 to l o 3
Fig. 10. Schematic display of average error rates of some reactions of replication and gene expression. The error rates given are averages of in-vitro measurements;
error rates measured for a particular enzyme and a particular substrate may deviate considerably. The corresponding in-vivo error rates may also deviate considerably.
rates by a factor of = 100 to 1000 may be ascribed to the
proofreading, resulting in an overall error frequency for
the peptide bond formation of = 1 : lo4 to 1 : 105.[157-1591
The proofreading system of the ribosomes can be blocked
by antibiotics, leading to increased error rates and a loss of
GTP
Furthermore, different ribosomal mutants exist which allow the binding of incorrect aminoacyltRNAs or inaccurate peptide bond formation to occur owing to mutations in ribosomal proteins.['61,'621Ho wever,
nothing is known yet about the structural changes of the
ribosomal proteins induced by the binding of antibiotics o r
by specific mutations.
7. Outlook on Solved and Unsolved Problems
In the absence of an efficient proofreading mechanism,
D N A polymerases would work with error rates of = 1 : lo4
to 1 :10'; aminoacyl-tRNA synthetases, which have to discriminate between very similar amino acids, would work
with error rates of = 1 : 10' to 1 : lo2, and the ribosomal
protein synthesis apparatus would work with error rates of
= l : l o ' to 1 : l o 2 (Fig. 10). The proofreading systems
lower the error rates of prokaryotic D N A replication to
= 1 :lo' to 1 :lo", and in eukaryotic cells even to
= 1 : 10". All reactions involved in the expression of the
genetic information show error rates of = 1 : lo3 to 1 : lo5.
The overall error rate of protein biosynthesis, 1 :3000, determined by Loftfield["] in 1963, has been confirmed in
different system^."^^-'^^^ The latest data seem to indicate
an additional dependence of the error rates on the speed of
protein synthesis and on the codon U S ~ ~ . I ~ ~
In all cases examined so far, the energy-dependent correction systems lead to a reduction of error rates by a factor of = 100 to 1000. If an even higher factor is necessary,
an additional system, requiring further energy, has to be
established, like the postreplicative proofreading of D N A
replication.
Angew. Chem. Inr. Ed. Engl. 24 (1985) 1015-1025
The basic mechanisms accounting for the extremely high
accuracy of D N A replication and protein biosynthesis
were phenomenologically discovered, but there is still a
lack of information about the chemistry of these mechanisms. Preliminary results of the X-ray analysis of a tryptic
fragment of methionyl-tRNA synthetase (E. coli) and of tyrosyl-tRNA synthetase (Bacillus stearothermophilus) have
been published.['70.I 7 l 1 In both structures, the shape of the
amino acid binding site is clearly visible, but the carboxy
terminus of nearly 100 amino acids is either missing or disordered in the electron density map. Although a high degree of homology in the amino acid binding sites of these
aminoacyl-tRNA synthetases is present, the correlation of
amino acids with the correction systems is still impossible.
Deeper insight is expected from "site-directed mutagenesis"
such as those already carried out with
tyrosyl-tRNA ~ y n t h e t a s e . [ ' ~However,
~I
the tRNA binding
site and the mechanism of transfer of the aminoacyl residue to the tRNA remain unclear due to the lack of the carboxy terminus in the X-ray structure^.['^^.'^^] For tRNAPhe
and tRNAAsp, however, the enzyme-binding regions have
been characterized thoroughly.[1761
All experimental examinations and theoretical considerations of error rates of enzymes lead to the following principle: sufficient accuracy is not possible without energy input; infinite accuracy would require infinite amounts of
energy. Data measured so far, showing an economic compromise between these extremes, correlate increasingly
better with detailed mathematical models.[63.177-1801 The
flux of components, energy, and information has many
~ - characteristics
~ ~ ~ ~
of a system that works far from chemical
The authors wish to thank Professor Dr. R . B. Lofttfield
and Dr. J . Taylor for critically reading the manuscript.
Received: August 23, 1984 [A 556 IE]
German version: Angew. G e m . 97 (1985) 1033
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