©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Proofreading in Vivo
EDITING OF HOMOCYSTEINE BY AMINOACYL-tRNA SYNTHETASES IN ESCHERICHIA COLI(*)

(Received for publication, May 25, 1995)

Hieronim Jakubowski

From the Department of Microbiology and Molecular Genetics, University of Medicine and Dentistry- New Jersey Medical School, Newark, New Jersey 07103

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

In many cases differences in intrinsic binding energies of amino acids to aminoacyl-tRNA synthetases (AARS)()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) ).

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,()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).


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).


MATERIALS AND METHODS

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.


RESULTS AND DISCUSSION

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.

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.


FOOTNOTES

*
This work was supported by American Cancer Society Grant NP-904 and National Science Foundation Grant MCB-9218358. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The abbreviations used are: AARS, aminoacyl-tRNA synthetase(s); e.g. Ile-RS, isoleucyl-tRNA synthetase, etc.; pIle-RS denotes plasmid or strain overproducing Ile-RS, etc.

H. Jakubowski, unpublished data.


ACKNOWLEDGEMENTS

I thank Sylvain Blanquet, Charles Carter, Gilbert Eriani, Michael Hrtlein, Paul Schimmel, and Ira Schwartz for plasmids.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.