In the following paragraphs, we summarize the findings on the crucial nucleotides and amino acids involved in mutual interactions that were achieved using our computer models. Further, we present here the results of extensive MD simulations of 18 complexes covering all possible combinations of six σ4 segments (see Table 2), and three −35 elements derived from our four promoters (σD-dependent PrsdA and synthetic hybrid promoter PD35-rsdAH10-uvrD3 share the same −35 element—see Table 1). Finally, we will provide tentative explanations of the observed biological activities (see Fig. 3).

Our computer models (Figs. 4, 5) show that interactions between amino acids and nucleotides can be divided into sequence-specific, which involve nucleobases, and sequence non-specific, which involve negatively charged sugar-phosphate backbone of DNA attracting long and flexible side-chains of basic amino acids (arginines or lysines). Further, amino acids with hydroxyl groups that can form hydrogen bonds (for example Y155 or T165) are also involved.

Fig. 4

Domain 4.2 of the σH subunit interacting with the −35 element of the PuvrD3 promoter. Amino acids from σH that were mutated in this study have red labels. Labels of amino acids that significantly interacted with the promoter in MD simulations are highlighted in bold. For clarity, amino acids are shown separately: (left) mutated only in 6aa mutants; (middle) mutated in all cases; (right) amino acid E140, which was mutated in the 4aa_K and 6aa_K mutants to compensate for the R172 to A172 mutation. I.e. so that a positively charged amino acid side chain is present at the site that could interact with the negatively charged backbone of the nucleic acid. In addition, R175, which interacts with the nucleotide base at position −35, as well as the basic amino acids K178, R171, R177, and K156, which are important for interaction with the sugar-phosphate backbone of the nucleic acids, are shown here

Fig. 5

Domain 4.2 of the σD subunit interacting with the −35 element of the PrsdA promoter. Amino acids from σD that were mutated have blue labels. Labels of amino acids that appeared to be particularly important in MD simulations are highlighted in bold. For clarity, amino acids are shown separately: (left) mutated only in 6aa mutants; (middle) mutated in all cases; (right) K140 from the 4aa_K and 6aa_K mutants that compensates for the R172 to A172 mutation. Also shown is the crucial R175 interacting with the nucleotide base at position −35 and the basic amino acids R181 and R148, which are important for interaction with the sugar phosphate backbone of the nucleic acids

Nucleotides involved in σ4 region–promoter interactions

In the promoters, Pcg0441, PuvrD3, PrsdA/PD35-rsdAH10-uvrD3, the non-conserved bases at the first and second (i.e. −35, −34) positions within the −35 elements of the non-template DNA strand (CTAAC/GGAAT/GTAAC)and the non-conserved base at the fifth (i.e. −31) position of the opposite (template) DNA strand (GATTG/CCTTA/CATTG) were found to participate in the most important sequence-specific interactions with polar amino acids of σ4 subunits (Figs. 4, 5).

Specifically, the C−35ntT−34nt–G−31t bases within the σD35H10-dependent promoter Pcg0441, are involved in these contacts. In the case of the σH-dependent promoter PuvrD3, the G−35ntG−34nt-A−31t bases are involved. Finally, in the case of the σD-dependent PrsdA, and the hybrid promoter PD35-rsdAH10-uvrD3, the G−35ntT−34nt–G−31t bases are involved (Figs. 4, 5).

Furthermore, the conserved bases −33, −32 (more specifically AA/TT in the non-template/template DNA chain) are in close contact with the σ4.2H/σ4.2D helix between the mutated amino acids at positions 170 and 171 (i.e. M170, S171 or R170, V171). The methyl groups of both T bases participate in stabilizing hydrophobic interactions, especially with the bulky non-polar side-chain of V171.

Moreover, several nucleotides surrounding the canonical −35 element may be in at least occasional contact with mutated amino acids. This may to some extent modulate the recognition between specific σ-subunits and promoters. For example, the −30 nucleotide interacts with the amino acid R170 or M170, or the −37, and −36 nucleotides interact with the amino acid K140.

Amino acids involved in σ4–promoter interactions

The central helix of the σ4.2H/σ4.2D segments is involved in interactions with the −35 elements of promoters.

The G/A base at position −31 of the template strand of DNA interacts with R170 at σD. In particular, if G is in position −31, up to two stabilizing hydrogen bonds may be formed. Alternatively, the long flexible side chain of R170 can create the so-called salt bridge with negatively charged phosphate groups of the sugar–phosphate backbone of DNA.

In the case of σH, the G/A base at position −31 interacts with M170. Methionine cannot form hydrogen bonds with bases or salt bridges with phosphate groups of the sugar–phosphate backbone of DNA like R170. However, the M170 side chain forms a tight cluster with the side chains of amino acids L166, Y155, and R177 (not present in σD). The latter two amino acids interact with the sugar–phosphate backbone of DNA. This seems to be how the presence of an either optimal or suboptimal interaction partner i.e. A−31 or G−31 in the vicinity of M170 can result in either stabilization or destabilization of the σ–promoter complex.

In the case of mutant σD35H10 subunits, a specific situation occurs (Fig. 6). There is R170 transferred from σD, but there are also amino acids L166, Y155, and R177 from σH. In our MD simulations, it took usually quite a while before long side chains of R170 and Y155 relaxed and settled properly into an arrangement without steric conflicts/clashes between them.

Fig. 6

In the case of mutant σD35H10 subunits (bottom), a specific situation occurs. There is R170 transferred from σD (top right) but there is also amino acid Y155 from σH (top left). In MD simulations, it took quite a while before the long side chains of R170 and Y155 relaxed and settled properly into an arrangement without steric clashes between them. More specifically, the optimal interaction of R170 with base −31 was mostly observed when spatially close Y155 was not bound to the phosphate group of DNA. Steric conflicts between R170 and Y155 could be one of the reasons, why we generally observed weaker transcription with the σD35H10 mutants than was achieved with σD that lacks Y155

The G/T bases at position −34 of the non-template DNA strand interact with amino acids that are specific for σH (S171) and σD (V171) subunits (Fig. 7). While S171 forms stabilizing hydrogen bonds with the G–34 base, the attraction of V171 and T–34 stems from their hydrophobicity.

Fig. 7

The G/T bases at position −34 of the non-template DNA strand interact with amino acids that are specific for σH (S171] and σD (V171) subunits. While S171 forms stabilizing hydrogen bonds with the G−34 base, the attraction of V171 and T−34 stems from their hydrophobicity

Close contacts of atypical interacting partners S171 and T−34 (in σH complexes with the σD-dependent promoter PrsdA, natural D35H10 hybrid promoter Pcg0441, and artificial hybrid promoter PD35-rsdAH10-uvrD3) are destabilizing. This is because a hydrogen bond formed between the hydroxyl group of S171 and oxygen atom O4 of T–34 leads ultimately to the disruption of other stabilizing interactions.

In contrast, close contacts of atypical interacting partners V171 and G–34 in complexes of σD and σD35H10 mutants (σH_4aa, σH_4aa_K, σH_6aa, and σH_6aa_K) with the σH-dependent promoter PuvrD3, don't seem to be destabilizing.

Base G at the position −35 of the non-template DNA strand of either σH-dependent PuvrD3 or σD-dependent PrsdA interacts (via two hydrogen bonds involving atoms O6 and N7) with an arginine side chain R175 (Fig. 8), which is present in the equivalent spatial position of both σH and σD subunits. These hydrogen bonds with the involvement of R175 (conserved in σH and σD) we consider to be the most important. After their disruption, a substantial repositioning of the DNA:DNA duplex relative to σ4 domains usually followed quickly.

Fig. 8

(Left) Base G at the position −35 of the non-template DNA strand of either σH-dependent PuvrD3 or σD-dependent PrsdA interacts (via two hydrogen bonds involving atoms O6 and N7) with an arginine side chain R175, which is present in the equivalent spatial position of both σH and σD subunits. (Right) The C base at the first position of the Pcg0441 −35 element does not allow usual hydrogen bonding with the R175 side chain. Instead, this R175 side chain interacts with the guanine base at position −35 in the complementary template strand of DNA

As regards sequence non-specific interactions, R181 of both σ4H/σD domains interact with the negatively charged sugar–phosphate backbone of DNA chains and provide further stabilization of σ4–promotor complexes. Further, there is another basic amino acid K178, which is present only in σ4H and in its mutants.

Stability of σ4D–promoter complexes in MD simulations

Let us summarize the results of 800 ns MD simulations for 18 simulated systems grouped based on the involved σ4 subunits.

Interactions of σ4H with promoters

As expected, σ4H created the most stable complex with the σH-dependent promoter PuvrD3. All possible stabilizing interactions mentioned above were found: sequence-specific (G–35–R175, G–34–S171, A−31–M170) as well as sequence non-specific (i.e. mediated by binding of side chains of Y155, R177, K156, R172, T168, R181, K178 to the sugar–phosphate backbone of nucleic acids).

On the other hand, in σD- and σD/σH-dependent promoters (i.e. PrsdA, Pcg0441, and PD35-rsdAH10-uvrD3), most of these interactions were interrupted, the orientation of the DNA:DNA duplex relative to σ4H was significantly changed, or even this complex was completely abrogated. In any case, hydrogen bonding between undue partners (S171 and T–34) was a primary destabilizing factor.

Interactions of σ4D with promoters

All complexes were quite stable and only small deviations were observed for σ4D, expected stabilizing interactions mentioned above were observed: sequence-specific (C/G–35–R175, T–34–V171, G–31–R170) as well as sequence non-specific (i.e. mediated by binding of side chains of R148, E156, K140, T165, R181 to the sugar phosphate backbone of nucleic acids).

The only exception was the σH-dependent promoter PuvrD3, where significantly greater disturbance of interactions was transiently observed, and it is questionable whether this complex would remain stable during a longer MD simulation.

Interactions of σ4H mutants with promoters

Mutations of only 4–7 amino acids that bring σH closer towards σD (resulting in σH_4aa, σH_6aa, σH_4aa_K, or σH_6aa_K constructs) were sufficient for the stabilization of complexes of all mutated σH subunits with σD- and σD/σH-dependent promoters (PrsdA, Pcg0441 and PD35-rsdAH10-uvrD3).

On the contrary, due to these mutations, certain destabilization of complexes with the σH-dependent PuvrD3 promoter occurred. Here, the most stable was the complex with σH_6aa_K,

The optimal interaction of R170 with base -31 was mostly observed when spatially close Y155 was not bound to the phosphate group of DNA. Steric conflicts between those two amino acids could be one of the reasons, why we generally observed weaker transcription with the σD35H10 mutants than was achieved with σD that lacks Y155.