(Received for publication, May 25, 1995)
From the
Editing reactions are an essential part of biological
information transfer processes that require high accuracy, such as
replication, transcription, and translation. The editing in amino acid
selection for protein synthesis by an aminoacyl-tRNA synthetase, the
first proofreading process discovered in the flow of genetic
information, prevents attachment of incorrect amino acids to tRNA. Of
numerous editing reactions studied in vitro, only one, editing
of homocysteine by methionyl-tRNA synthetase, has also been
demonstrated in vivo. It is therefore unclear to what extent
editing of errors is physiologically relevant. Here we show that
isoleucyl- and leucyl-tRNA synthetases also edit homocysteine by
cyclizing it to homocysteine thiolactone in the bacterium Escherichia coli. These and other data also suggest that
metabolite compartmentation or channeling governs which synthetase
participates in editing in bacterial cells. In many cases differences in intrinsic binding energies of amino
acids to aminoacyl-tRNA synthetases (AARS) The non-protein amino acid
homocysteine (Hcy), an obligatory precursor of methionine in all cells,
poses an accuracy problem for the protein biosynthetic apparatus. Hcy
is misactivated in vitro by three AARS, Met-RS,
Ile-RS(4) , and Leu-RS(5) , at a frequency exceeding
the frequency of translational errors in vivo. Two other
synthetases, Val-RS (4, 6) and Lys-RS,
Figure S1:
Scheme 1
So far, only editing of Hcy by Met-RS has
been shown to be a physiologically important process that prevents
misincorporation of Hcy into tRNA and protein in Escherichia
coli(9) , yeast (10) , and some mammalian
cells(11) . It is unclear whether other editing reactions
that have been demonstrated in vitro are physiologically
relevant. Our previous data indicated that, because of compartmentation
of Hcy metabolism in E. coli(12) , endogenous Hcy (formed in the methionine biosynthetic pathway) is edited
exclusively by Met-RS. To test whether exogenous Hcy (taken up
from the medium) can be edited by other aminoacyl-tRNA synthetases,
cultures of E. coli cells that overproduce individual
aminoacyl-tRNA synthetases have been incubated with Hcy and assayed for
Hcy thiolactone by UV spectrometry and thin layer chromatography (TLC).
The present
discovery that Ile-RS and Leu-RS, in addition to Met-RS, are
responsible for editing of exogenous (taken up from the
medium) Hcy does not contradict the original observation that Met-RS is
exclusively involved in editing of endogenous (formed in the
methionine biosynthetic pathway) Hcy(9, 12) . Taken
together, results of the present and previous experiments demonstrate
that endogenous Hcy is accessible only to, and edited only by,
Met-RS. On the other hand, exogenous Hcy is accessible to, and
edited by, any synthetase that is able to misactivate it efficiently.
This further indicates that the extent of editing of Hcy in bacterial
cells is governed by compartmentation or channeling of Hcy metabolism
that may limit access of some synthetases to a particular intracellular
amino acid pool.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)are
inadequate to give the required accuracy of translation. This has
necessitated the evolution of a second determinant of specificity,
proofreading or editing mechanisms that involve the expenditure of
energy to remove errors in amino acid selection (reviewed in (1, 2, 3) ).
(
)misactivate Hcy less efficiently. These five enzymes
possess an efficient editing mechanism that destroys the Hcy-AMP
intermediate and prevents misincorporation of Hcy into
tRNA(4, 8) . The editing reaction involves
nucleophilic attack of the side chain thiolate group of Hcy on its
activated carboxyl group. A cyclic thioester, Hcy thiolactone, is a
product of these editing
reactions(4, 5, 6)
(Fig. S1).
Plasmids and Host Strain
Plasmids overexpressing
AARS were obtained from the following sources: pIle-RS from P.
Schimmel(13) ; pLeu-RS, pSer-RS, pAsn-RS from M.
Hrtlein(14, 15, 16) ;
pMet-RS, pVal-RS, pLys-RS from S. Blanquet(17) ; pArg-RS,
pAsp-RS, pCys-RS, pPro-RS from G.
Eriani(18, 19, 20, 21) ; pPhe-RS
from I. Schwartz; pTrp-RS from C. Carter. Plasmids were overexpressed
in E. coli strain JM101(7) . Specific activity
measurements and SDS-polyacrylamide gel electrophoretic analysis of
proteins in crude bacterial extracts confirmed that synthetases were
overexpressed from plasmids to a level 40-70-fold greater that
the expression level from the corresponding chromosomal gene in JM101.
Intracellular concentration of AARS in the plasmid-bearing strains was
estimated to be 0.5-1.3 mM(17) .Spectroscopic Assay for Homocysteine
Thiolactone
Bacterial cultures (cell density of 4
10
cells/ml) were incubated with 5 mMDL-homocysteine in M9 medium. Cultures of plasmid-bearing
strains also contained 0.1 mg/ml ampicillin. Aliquots (0.075-0.75
ml) of cultures were clarified by microcentrifugation, the cell-free
medium was brought up to 0.75 ml with fresh medium where necessary, and
the spectrum from 220 to 300 nm of the cell-free medium was recorded
against fresh medium as a reference using a Hewlett-Packard diode array
spectrometer, model 8451A. The standard calibration curve was prepared
by A
measurements of known quantities of
homocysteine thiolactone in M9 medium. An A
= 0.35 was equivalent to 0.1 mM of the
thiolactone.
TLC Analysis
Aliquots (10 µl) of bacterial
cultures were applied on the origin line of a hard layer silica gel TLC
plate (Merck). The plate was developed with butanol:acetic acid:water
(4:1:1, v/v) as a solvent. Homocysteine thiolactone spots were
visualized under UV and also by staining with ninhydrin
(yellow)(4) . Homocysteine thiolactone at concentrations as low
as 0.1 mM can be detected in bacterial cultures using this TLC
system.
Overproducers of Ile-RS and Leu-RS Also Overproduce Hcy
Thiolactone
An absorption peak with a maximum at 240 nm,
characteristic of Hcy thiolactone, appeared in spectra of the media
from cultures of cells overexpressing Ile-RS and Met-RS after only 1 h
of incubation with Hcy. With pLeu-RS cultures, the A peak appeared after 5 h. Much less pronounced A
peaks appeared in the spectra of other cultures, including JM101
host cells culture, after 24 h of incubation. TLC confirmed that Hcy
thiolactone was formed in pIle-RS, pLeu-RS, and pMet-RS cultures after
5 h and in other cultures after 24 h of incubation. In all cases, a
UV-absorbing, base-sensitive spot that stained yellow with
ninhydrin(4) , comigrating with authentic Hcy thiolactone, was
detected after culture media samples were subjected to TLC.
Quantitation of Hcy thiolactone formed in bacterial cultures (Table 1) indicates that in overproducers of Ile-RS, Leu-RS, and
Met-RS, the rate of synthesis of Hcy thiolactone is up to 30-fold
greater than in the JM101 E. coli host. Overproducers of other
AARS such as Arg-RS, Asp-RS, Cys-RS, Lys-RS, Phe-RS, Pro-RS, Ser-RS,
Trp-RS, and Val-RS produced Hcy thiolactone at very low levels, similar
to that observed for the JM101 E. coli host strain (Table 1). No detectable (by UV absorption) Hcy thiolactone was
formed in the absence of bacterial cells or exogenous Hcy. It should be
noted that in the absence of exogenous Hcy, the thiolactone is
detectable in bacterial cultures labeled with
[
S]sulfate; under these conditions, Hcy
thiolactone is formed at a rate of about 0.1 µM/1
h(9) .
Plasmid-dependent Synthesis of Hcy Thiolactone Is
Inhibited by a Cognate Amino Acid
To show that increased Hcy
thiolactone synthesis in some overproducers of AARS is in fact due to a
particular synthetase, effects of cognate and noncognate amino acids on
the thiolactone synthesis were determined. As expected, in experiments
with the Ile-RS-, Leu-RS-, and Met-RS-overproducing strains,
90-97% inhibition of Hcy thiolactone synthesis was observed only
in the presence of a corresponding cognate amino acid (Table 2).
For example, synthesis of Hcy thiolactone in cultures overproducing
Ile-RS was 97% inhibited by isoleucine but not by methionine. The
inhibition was specific for Ile-RS; isoleucine did not significantly
inhibit Hcy thiolactone synthesis in Leu-RS- and Met-RS-overproducing
cultures. For unknown reasons, isoleucine stimulated Hcy thiolactone
synthesis in the Met-RS overproducer. Lysine and cysteine, used as
controls, did not significantly affect Hcy thiolactone synthesis in any
of the tested cultures (Table 2). Thus, the high levels of Hcy
thiolactone synthesis in Ile-RS-, Leu-RS-, and Met-RS-overproducing
cultures are due to a particular synthetase that was overproduced.
In a Wild Type Bacterium Exogenous Hcy Is Cyclized to the
Thiolactone by Three Synthetases, Ile-RS, Leu-RS, and
Met-RS
Attempts to determine contribution by each synthetase to
Hcy editing in the wild type JM101 strain were also made. Due to the
relatively low ratio of signal to noise observed with this strain, only
qualitative assessments by TLC were possible. The low level of Hcy
thiolactone produced by JM101 was abolished when isoleucine, leucine,
and methionine were simultaneously added to the culture. Either amino
acid alone partially inhibited the synthesis of Hcy thiolactone by
JM101. This indicates that Ile-RS, Leu-RS, and Met-RS, expressed from
the chromosome in JM101, contribute about equally to editing of
exogenous Hcy in bacterial cells. Val-RS and Lys-RS that edit Hcy in vitro (albeit much less efficiently than Ile-RS, Met-RS,
and Leu-RS) do not seem to contribute significantly to editing in
vivo, even when overexpressed. Thus, for AARS that exhibit
inadequate initial selectivity, editing of errors in amino acid
selection is a physiologically important function.
I thank Sylvain Blanquet, Charles Carter, Gilbert
Eriani, Michael Hrtlein, Paul Schimmel, and Ira
Schwartz for plasmids.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.