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Journal of Bacteriology, March 2007, p. 2174-2175, Vol. 189, No. 5
0021-9193/07/$08.00+0     doi:10.1128/JB.01462-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

"Cold-Sensitive" Mutants of the Lac Repressor{triangledown}

Andrew Barker,1,2 Stefan Oehler,1,3* and Benno Müller-Hill1

Institut für Genetik der Universität zu Köln, Zülpicherstr. 47, D-50674 Köln, Germany,1 Western Australian Institute for Medical Research, Rear 50 Murray Street, Perth, WA 6000, Australia,2 IMBB-FORTH, Vassilika Vouton, P.O. Box 1378, GR-71110 Heraklion, Crete, Greece3

Received 15 September 2006/ Accepted 6 December 2006


   ABSTRACT
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Thirteen of more than 4,000 single-amino-acid-replacement mutants of the Lac repressor, generated by suppression of amber nonsense mutants, were characterized as having a cold-sensitive phenotype. However, when expressed as missense mutations, none of the replacements cause cold sensitivity, implicating the suppression mechanism as being responsible for this phenotype.


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The Lac repressor (LacR) is one of the best analyzed proteins. Its three-dimensional structure is known at high resolution (4, 10). More than 4,000 single-amino-acid-replacement mutants have been analyzed (6, 11). Among these, 13 cold-sensitive mutants were found (Fig. 1). The single most striking feature of these mutants is their distribution: 11 out of 13 amino acid replacements in six out of eight codons (of a total data set of 4,042 single substitutions covering residues 2 to 329 of the 360-amino-acid long LacR) are located in the DNA-binding headpiece (11). These amino acid replacements have been generated by in vivo suppression of amber nonsense mutants using suppressor tRNAs of the indicated specificity (11). This clustering seems to suggest that in these cold-sensitive mutants, regions in the DNA binding domain of the Lac repressor "freeze" at low temperatures into stable structures which are unable to bind properly to the lac operator (1).


Figure 1
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  		FIG. 1. Cold-sensitive missense suppression variants of LacR presented by Miller and coworkers (11, 16). The protein sequence of the N terminus of LacR plus the two residues of the core protein for which there are cold-sensitive amino acid replacements are shown. Positions where replacements (indicated above the sequence) can lead to a cold-sensitive phenotype are in boldface. The residues of the DNA binding recognition helix (residues 17 to 26) are in italic. The indicated replacements were generated by suppression of an amber mutation at the respective position of an episomally encoded lacIq allele, using a suppressor tRNA of the indicated specificity (6, 11). The lacZ reporter was also episomally encoded in these strains.

 
Interested in the biochemical properties of these mutants, we introduced appropriate missense mutations into plasmid pWB1000, which constitutively produces about 400 times the lacI+ amount of wild-type (tetrameric) LacR (8, 9). In order to verify the cold-sensitive nature of the introduced mutations, the ability of each mutant protein to repress a chromosomal lacIO+p+Z+Y+ allele in trans was tested at 37°C and 25°C. Repression values and induction with the gratuitous inducer isopropyl-ß-D-thiogalactopyranoside (IPTG) were determined by ß-galactosidase assays of the relevant strains (6, 8, 11, 12, 13). We were surprised to see only borderline cold-sensitive phenotypes (Table 1), with a maximum twofold-greater repression at 37°C than at 25°C for S16C, V20C, V23A, H29A, H29E, K33H, K33F, and K33E. Some alleles even show heat sensitivity, with a 12-fold-greater repression for R35E or a threefold-greater repression for E259F at 25°C.


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  		TABLE 1. In vivo activities of missense LacR mutants at 25°C and 37°Ca

 
Since overexpression of LacR might compensate for cold sensitivity, a series of strains was constructed, in which each of the cold-sensitive LacR variants is produced at the wild-type level (about 10 tetramers per cell). The reporter system used consists of a chromosomal copy of lacZ under control of a lacI gene integrated at the attachment site for bacteriophage {lambda} (15). Repression values were determined by comparing lacZ expression in the presence or absence of the gratuitous inducer IPTG at 37°C or 25°C to expression in the absence of any LacR. Here, only one mutation, S16A, shows a borderline cold sensitivity, with about 1.6-fold-weaker repression at 25°C than at 37°C. R35E retains a clear heat-sensitive phenotype, with a roughly eightfold-reduced repression at 37°C (in addition to being substantially reduced in its ability to repress even at 25°C), while V23C and H29E (both with 1.9-fold-reduced repression) and K33F (with 1.6-fold-reduced repression) show weak heat sensitivity. All mutants are fully inducible at both 25°C and 37°C, insofar as it is possible to measure induction of the essentially null mutant E259F (data not shown). Thus, not only are none of the 13 alleles cold sensitive, but neither V150C nor E259F has a LacIs phenotype, contrary to earlier reports (11, 14, 16).

We also tested a subset of the mutations for cold sensitivity in vitro. Partially purified extracts containing LacR alleles S16A, V20C, H29A, and K33E were prepared, and their ability to bind lacOid-containing plasmid DNA was compared to that of wild-type LacR in equilibrium binding studies (1, 3). No differences were detected in filter-binding experiments at 4°C, 25°C, or 37°C (data not shown).

These unexpected results prompted us to verify the results obtained by Miller and coworkers (11, 16). Strains bearing the relevant lacI(Am) nonsense mutations and suppressors were obtained from Miller, and their identities were confirmed by DNA sequencing. We were largely able to reproduce the results of Miller and coworkers (data not shown).

It thus appears that there are no cold-sensitive mutants of LacR. Given the wealth of characterized amino acid replacements for all positions of the headpiece and core protein, it seems justified to assume that no single amino acid replacement can turn the Lac repressor into a cold-sensitive protein.

There are, on the other hand, well-documented cases of true cold-sensitive mutants of other proteins (5, 7). The case of the Lac repressor should be helpful in determining which features of a protein make it amenable to cold sensitivity.

One question remains to be answered: how does the apparent cold sensitivity of the suppressed nonsense mutants arise? Wild-type Lac repressor has restarts at codons GUG23, GUG38, AUG42, and UUG62. Several of these have been removed in some alleles used by Miller et al., but all alleles retain the restart at position 42 (6). Suppression of nonsense codons leads to the expression of a certain fraction of full-length protein, while some to most ribosomes still stop but can reinitiate translation at a downstream start codon. Restart products of Lac repressor core protein which lack the DNA binding domain will form inactive mixed oligomers with the full-length repressor and thus be dominant negative when expressed in sufficient amounts. One could argue that their concentration increases at low temperature. However, this is obviously not the full explanation. Why are there cold-sensitive nonsense mutations at positions 16 and 20 but not at positions 17, 18, 19, and so on? The cold-sensitive mutants of the Lac repressor remain, on a different level, an enigma.


   ACKNOWLEDGMENTS
 
We are grateful to Jeffrey Miller and to Tony Poteete for their generous provision of materials used and to Jeffrey Miller for commenting on an earlier version of the manuscript. We thank Karin Schnetz for providing laboratory space.

This work was supported by grants from the Deutsche Forschungsgemeinschaft and Fonds der Chemie.


   FOOTNOTES
 
* Corresponding author. Mailing address: IMBB-FORTH, Vassilika Vouton, P.O. Box 1378, GR-71110 Heraklion, Crete, Greece. Phone: 30 2810-391144. Fax: 30 2810-391104. E-mail: oehler{at}imbb.forth.gr. Back

{triangledown} Published ahead of print on 15 December 2006. Back


   REFERENCES
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  6. Kleina, L. G., and J. H. Miller. 1990. Genetic studies of the lac repressor. XIII. Extensive amino acid replacements generated by the use of natural and synthetic nonsense supressors. J. Mol. Biol. 212:295-318.[CrossRef][Medline]
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Journal of Bacteriology, March 2007, p. 2174-2175, Vol. 189, No. 5
0021-9193/07/$08.00+0     doi:10.1128/JB.01462-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.




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