Department of Life Sciences, Ben-Gurion University of the Negev, Beer-Sheva, 84105, Israel1
Author for correspondence: Zeev Barak. Tel: +972 8 6461713. Fax: +972 8 6461710. e-mail: barakz{at}bgumail.bgu.ac.il
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ABSTRACT |
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Keywords: acetohydroxyacid synthase, acetolactate synthase, branched-chain amino acids, sulfonylurea, amino acid biosynthesis
Abbreviations: AHAS, acetohydroxyacid synthase; ILHX, isoleucine hydroxamate; SMM, sulfometuron methyl
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INTRODUCTION |
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To study the translational frameshifting mechanism, Gallant & Lindsley (1992) developed a bacterial system in which an original shifty region to be studied is inserted after the lac promoter and the translational initiation codon AUG and followed by an out-of-frame lacZ reporter gene. As the reporter gene is in-frame with the expected frameshift, the system allows quantitative determination of translational frameshifting by assay of the reporter gene product, ß-galactosidase. The sequence TTTCAAG led to a -1 translational frameshifting under conditions of starvation for charged tRNALys as a result of ribosome stalling at the lysyl codon, AAG, and frameshifting was correlated directly with the level of starvation (Gallant & Lindsley, 1992
). Changing the codon to the isoleucine-encoding sequences, ATA or ATC, retained the translational frameshifting properties of the system, except that starvation for charged tRNAIle was required to initiate frameshifting (Barak et al., 1996
). In each case, starvation was achieved by specific inhibition of aminoacyl-tRNA charging by the hydroxamate analogue of the amino acid. In principle, translational frameshifting should also be caused by a deficiency of an amino acid whose codon is in the shifty region, i.e. by inhibition of the biosynthesis of the amino acid.
In this study we demonstrate the use of translational frameshifting as a tool to estimate the degree of starvation for isoleucine in Salmonella typhimurium, when branched-chain amino acid biosynthesis is specifically inhibited by SMM.
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METHODS |
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Bacterial strain, growth and media.
Salmonella typhimurium LT2-TV105 (LaRossa & Schloss, 1984 ) expresses only isozyme II of the three AHAS isozymes of the enteric bacteria (Fig. 1
). This isozyme is strongly inhibited by the herbicide SMM and is resistant to valine inhibition (LaRossa & Schloss, 1984
; Schloss et al., 1988
).
Plasmid pMLB1115 with the shifty sequence TTTCATA (Barak et al., 1996 ) was designed for -1 translational frameshifting measurements at the isoleucine codon by means of the ß-galactosidase reporter gene. A strain of S. typhimurium TV105 carrying this plasmid was isolated after transformation by electroporation (Pfan & Youderian, 1990
; Takedo, 1988
). The transformant was selected to be resistant to kanamycin and ampicillin coded by the host genome and the plasmid, respectively.
S. typhimurium TV105(pMLB1115) was grown in minimal medium M63 (Miller, 1972b ) containing kanamycin (50 mg l-1) and ampicillin (150 mg l-1). Cultures were grown with shaking (200 r.p.m.) in a gyratory water bath at 37 °C. Growth was followed by measuring turbidity with a KlettSummerson colorimeter using a 660 nm filter.
Determination of translational frameshifting.
Translational frameshifting was measured in starved and control cultures by determination of the amount of ß-galactosidase produced after induction with IPTG (2 mM) and cAMP (2·5 mM), in one bacterial generation. The concentrations of the inducers were optimized to give maximal induction without interference with bacterial growth rate (data not shown). ß-Galactosidase activity was measured as described by Miller (1972a ). Protein concentrations were determined by the Bradford method (Bradford, 1976
) using the Bio-Rad Protein Assay Kit with bovine serum albumin as a standard.
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RESULTS AND DISCUSSION |
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 5 July 2001;
revised 29 October 2001;
accepted 16 November 2001.
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