Correlating protein footprinting with mutational analysis in the bacterial transcription factor sigma54 (sigmaN)

Abstract

Protein footprints of the enhancer-dependent sigma54 protein, upon binding the Escherichia coli RNA polymerase core enzyme or upon forming closed promoter complexes, identified surface-exposed residues in sigma54 of potential functional importance at the interface between sigma54 and core RNA polymerases (RNAP) or DNA. We have now characterised alanine and glycine substitution mutants at several of these positions. Properties of the mutant sigma54s correlate protein footprints to activity. Some mutants show elevated DNA binding suggesting that promoter binding by holoenzyme may be limited to enable normal functioning. One such mutant (F318A) within the DNA binding domain of sigma54 shows a changed interaction with the promoter regulatory region implicated in transcription silencing and fails to silence transcription in vitro. It appears specifically defective in preferentially binding to a repressive DNA structure believed to restrict RNA polymerase isomerisation and is largely intact for activator responsiveness. Two mutants, one in the regulatory region I and the other within core interacting sequences of sigma54, failed to stably bind the activator in the presence of ADP-aluminium fluoride, an analogue of ATP in the transition state for hydrolysis. Overall, the data presented describe a collection sigma54 mutants that have escaped previous analysis and display an array of properties which allows the role of surface-exposed residues in the regulation of open complex formation and promoter DNA binding to be better understood. Their properties support the view that the interface between sigma54 and core RNAP is functionally specialised.

Figure 1

Domain organisation of the K.pneumoniae σ⁵⁴. (A) The N-terminal 56 residues (region I) have roles in activator responsiveness. The major DNA binding determinants of σ⁵⁴ are localised in region III which contains a patch of 18 amino acids (residues 329–346) that UV-crosslink to DNA (1), a putative helix–turn–helix (HTH) motif (residues 366–386) that has been implicated in interacting with the start site proximal promoter element (the –12 region) (16,42) and a σ⁵⁴ characteristic motif of 10 amino acids (residues 454–463) known as the RpoN box that has been implicated in interacting with start site distal promoter element (the –24 region) (4,43). Residues 120–215 in region III of σ⁵⁴ contain the major core RNAP binding determinant (23). (B) Summary of protease-sensitive sites (12). The black bar indicates core RNAP-specific protection and dotted bars show closed complex-specific protection. (C) Sites where single mutations were introduced at surface-exposed positions.

Figure 2

In vivo activity and immunoblot of cell extracts prepared from K.pneumoniae UNF2792 cells containing mutant plasmids.

Figure 3

Escherichia coli core RNAP binding by purified σ⁵⁴ mutants. Holoenzymes (Eσ⁵⁴) were formed at 1:1 (lanes 2, 7, 12, 18, 23, 28, 33 and 39), 1:2 (lanes 3, 8, 13, 19, 24, 29, 34 and 40), 1:4 (lanes 4, 9, 14, 20, 25, 30, 35 and 41) and 1:8 (lanes 5, 10, 15, 21, 26, 31, 36 and 42) ratios of core RNAP (250 nM) to σ⁵⁴. Free core RNAP (lanes 1, 17 and 38) and free σ⁵⁴:wild-type (lane 6), E36G (lane 11), F318A (lane 16), E325G (lane 22), E410A (lane 27), E414A (lane 32), E431A (lane 37) and EE410/414AA (lane 43) are also shown.

Figure 4

Homoduplex DNA binding. (A) Sinorhizobium meliloti nifH promoter probes used for the DNA binding assays (see text for details). The consensus GG and GC of the σ⁵⁴ binding sites are highlighted and the mismatched regions are boxed in black. (B) Binding of wild-type and mutant σ⁵⁴ proteins (at 1 µM) to homoduplex promoter DNA probe. (C) Titration of wild-type, F318A, E410A and EE414/410AA on the homoduplex promoter probe.

Figure 5

Binding (lanes 2, 4, 6, 8, 10, 12, 14 and 16) and isomerisation (lanes 3, 5, 7, 9, 11, 13, 15 and 17) of wild-type and mutant σ⁵⁴ in the presence of PspFΔHTH and dGTP on the early melted promoter probe. The amount of DNA bound (σ⁵⁴–DNA) and isomerised (ssσ⁵⁴–DNA) complexes formed are indicated as % DNA shifted and % isomerised, respectively.

Figure 6

σ⁵⁴ Holoenzyme (Eσ⁵⁴)-promoter–DNA interactions. (A) Binding of 100 nM mutant holoenzyme [formed with 1:4 core RNAP (E) to σ⁵⁴ ratio] to the homoduplex promoter DNA. (B) Stability of mutant holoenzyme on the early melted DNA probe following a 5 min heparin challenge. The percentage of Eσ⁵⁴–DNA complexes formed on both promoter probes are indicated in (A) and (B), respectively.

Figure 7

σ⁵⁴ Holoenzyme stability on the late melted promoter probe. (A) Binding (black bars) and stability (white bars) of wild-type and mutant σ⁵⁴ holoenzyme complexes formed on the late melted promoter probe following a 5 min heparin challenge. (B) Stability of activated wild-type and mutant closed complexes formed on the late melted probe following a 5 min heparin challenge.

Figure 7

σ⁵⁴ Holoenzyme stability on the late melted promoter probe. (A) Binding (black bars) and stability (white bars) of wild-type and mutant σ⁵⁴ holoenzyme complexes formed on the late melted promoter probe following a 5 min heparin challenge. (B) Stability of activated wild-type and mutant closed complexes formed on the late melted probe following a 5 min heparin challenge.

Figure 8

Nucleotide-dependent σ⁵⁴–activator complex formation. (A) Nucleotide-dependent binding of wild-type and mutant σ⁵⁴ proteins and (B) the holoenzymes thereof to ³²P-labelled PspFΔHTH. (C) Nucleotide-dependent binding of wild-type and mutant closed complexes formed on the late melted DNA probe to PspFΔHTH.

Figure 8

Nucleotide-dependent σ⁵⁴–activator complex formation. (A) Nucleotide-dependent binding of wild-type and mutant σ⁵⁴ proteins and (B) the holoenzymes thereof to ³²P-labelled PspFΔHTH. (C) Nucleotide-dependent binding of wild-type and mutant closed complexes formed on the late melted DNA probe to PspFΔHTH.

Figure 9

In vitro transcription from S.meliloti nifH promoter. (A) Activated transcription by wild-type and mutant holoenzymes at 37 and 15°C. The in vivo activity of the mutants are shown for direct comparison (see also Fig. 2). (B) Activator-independent ‘bypass’ transcription.

Figure 10

Fork junction DNA binding by F318A holoenzyme. (A) The fork junction probes used for the binding assays based on E.coli glnHp2 promotor. (B) Autoradiograph of binding of holoenzymes to the fork junction probes 1,2,3 and 4. The fraction of complexes formed with respect to wild-type σ⁵⁴ is shown for each fork junction probe.