Overlapping repressor binding sites regulate expression of the Methanococcus maripaludis glnK(1) operon

Abstract

The euryarchaeal transcriptional repressor NrpR regulates a variety of nitrogen assimilation genes by 2-oxoglutarate-reversible binding to conserved palindromic operators. The number and positioning of these operators varies among promoter regions of regulated genes, suggesting NrpR can bind in different patterns. Particularly intriguing is the contrast between the nif and glnK(1) promoter regions of Methanococcus maripaludis, where two operators are present but with different configurations. Here we study NrpR binding and regulation at the glnK(1) promoter, where the two operator sequences overlap and occur on opposite faces of the double helix. We find that both operators function in binding, with a dimer of NrpR binding simultaneously to each overlapping operator. We show in vivo that the first operator plays a primary role in regulation and the second operator plays an enhancing role. This is the first demonstration of overlapping operators functioning in Archaea.

Fig. 1. nif and glnK₁ promoter regions

A. nif promoter region. B. glnK₁ promoter region. TATA boxes (caps), transcription start sites (bent arrows), and operators (bold) are shown. Mutant glnK₁ operators are underlined.

Fig. 2. The glnK gene cluster

The location of the transcription start site is indicated by a bent arrow. The probe used for Northern blots is indicated by a short horizontal line. Primers used for RT-PCR are shown as arrows.

Fig. 3. Electrophoretic mobility shift analysis (EMSA) of NrpR binding to operator DNA

A. DNA probes containing operators shown in Fig. 1: wild type (O₁+O₂), O₂ only (O₁ mutant), O₁ only (O₂ mutant), and both operators mutant. No (-) or 120 nM (+) NrpR was mixed with the DNA. B. EMSA at different NrpR concentrations: O₁+O₂, 0, 0.05, 0.1, 0.15, 0.2, 0.4, 0.6, 1.0, and 1.5 nM; O₂ and O₁, 0, 0.25, 1, 2, 4, 8, 16, 32, and 64 nM. C. Plot of data from B.

Fig. 4. DNAse I footprinting analysis of NrpR binding to operator DNA

Footprints are delineated by bold lines and indicated with respect to the sequence. Double stranded DNA sequences on left and right represent O₁+O₂ and O₁ only, respectively. Boxed sequences, TATA box; bent arrow, transcription start site; inverted arrow pairs, operator(s); asterisks, hypersensitive sites. A, C and D. Lane 1, G+A ladder; lane 2, no NrpR; lane 3, 200 nM NrpR; lane 4, 400 nM NrpR. Units of DNAse I, 0.005 B. Lane 1, G+A ladder; lanes 2 and 3, no NrpR, lanes 4 and 5, 400 nM NrpR.; Units of DNAse I: lanes 2 and 4, 0.001; lanes 3 and 5, 0.002.

Fig. 5. EMSA of NrpR binding to nif and glnK₁ operators

DNA probes of equal length contained nif O₁ + O₂ or glnK₁ O₁ + O₂. Single and double arrows indicate shifts corresponding to NrpR binding to one or two operators respectively. NrpR concentrations were 0, 0.05, 0.1, and 2.0 nM.

Fig. 6. In vivo regulation of wild type and mutant glnK₁ promoter reporter fusions

Strains containing lacZ fusions to promoters with wild type (O₁ + O₂) and mutant (O₁ only, O₂ only, or both operators mutant) operators were grown under three nitrogen conditions as indicated, and β-galactosidase assays performed as described in Experimental Procedures.

Fig. 7. Models for the binding of NrpR to the nif and glnK₁ promoter regions

A dimer of dimers (tetramer) binds to the nif promoter region, which contains two operators centered three helical turns apart on the same face of the DNA helix. Two separate dimers bind on opposite sides of the helix in the glnK₁ promoter region, which contains two overlapping operators spaced only 6 bp apart.