Characterization of the dimerization domain in BglG, an RNA-binding transcriptional antiterminator from Escherichia coli

Abstract

The Escherichia coli transcriptional antiterminator protein BglG inhibits transcription termination of the bgl operon in response to the presence of beta-glucosides in the growth medium. BglG is an RNA-binding protein that recognizes a specific sequence partially overlapping the two terminators within the bgl transcript. The activity of BglG is determined by its dimeric state which is modulated by reversible phosphorylation. Thus, only the nonphosphorylated dimer binds to the RNA target site and allows readthrough of transcription. Genetic systems which test dimerization and antitermination in vivo were used to map and delimit the region which mediates BglG dimerization. We show that the last 104 residues of BglG are required for dimerization. Any attempt to shorten this region from the ends or to introduce internal deletions abolished the dimerization capacity of this region. A putative leucine zipper motif is located at the N terminus of this region. The role of the canonical leucines in dimerization was demonstrated by their substitution. Our results also suggest that the carboxy-terminal 70 residues, which follow the leucine zipper, contain another dimerization domain which does not resemble any known dimerization motif. Each of these two regions is necessary but not sufficient for dimerization. The BglG phosphorylation site, His208, resides at the junction of the two putative dimerization domains. Possible mechanisms by which the phosphorylation of BglG controls its dimerization and thus its activity are discussed.

FIG. 1

Generation of nested sets of deletions in bglG. pOAC101, which codes for a fusion between the DNA-binding domain of λ repressor and the entire bglG gene, was linearized with the indicated restriction enzymes and digested with BAL 31 nuclease for various times as described in Materials and Methods. (A) Shortening bglG from its 3′ end. (B) Shortening the second half of bglG from its 5′ end, i.e., from the HpaI site, located in the middle of the bglG gene towards the 3′ end of the gene. (C) Shortening the sequence encoding the 70 C-terminal residues of BglG from both ends.

FIG. 2

The putative leucine zipper in BglG. (A) Amino acid sequence of the leucine zipper motif found in BglG. The a and d positions of each heptad are shown. (B) Helical wheel representation of BglG leucine zipper. A total of 28 amino acids (residues Val¹⁷⁶ to Gln²⁰³) are included in the analysis, and the view is from the amino terminus. Positions around the helical wheel are labeled a through g. The positions of the canonical leucines in BglG are indicated.

FIG. 3

The canonical leucines in the leucine zipper motif of BglG are required for dimerization. The ability of BglG 104 C-terminal residues, wild-type and mutants with single or double leucine substitutions, to dimerize was deduced from their ability, when fused to λ repressor DNA-binding domain, to confer phage immunity and to repress λP_R-lacZ expression. Phage immunity was assayed by the spot test at phage concentrations ranging from 10⁹ to 10⁴ PFU/ml on lawns of the bacteria transformed with the indicated plasmids. Repression of the chromosomal λP_R-lacZ fusion was calculated from β-galactosidase (β-Gal) activities measured in cells grown in LB medium as described in Table 2, footnote d. The values (in Miller units) represent the averages of at least four independent measurements. The leucine substitutions are indicated by the large bold letters.