Transcription is the first and most regulated step in gene expression and is carried out by a single protein complex in bacteria, RNA polymerase. To coordinate changes with growth conditions and gene expression, RNAP often associates with other cellular factors, which can affect all steps in transcription from initiation to elongation and termination. Being the first step in gene expression, transcription initiation serves as the main target for hundreds of transcription factors. Most of them modulate the recruitment of RNAP to specific promoters, typically by promoting or hindering RNAP interactions with promoter elements.
DksA is a transcription factor in ¿- proteobacteria that is commonly associated with the stringent response, a universal bacterial stress response to starvation for carbon or amino acids. DksA has been shown to control initiation at many promoters but, unlike most regulators of transcription initiation, DksA does not recognize specific a DNA sequence and instead binds within the secondary channel of RNAP and modifies the enzyme’s properties using a yet-unknown mechanism. DksA, often in conjunction with an alarmone ppGpp, reduces the stability of RPO, the final streps in the formation on a catalytically active initiation complex, and alters transcription initiation at target promoters. pppGpp and DksA have been shown to act synergistically at many, but not all, promoters. In addition, ppGpp and DksA have been proposed to alter replication in the cell.
DksA belongs to a unique family of transcription factors which consists of GreA, GreB, Gfh1, Rnk1, and TraR. These factors share several features including a similar structure and binding site in the secondary channel of RNAP however, they carry out distinct functions in the cell.
Here we expanded the research on DksA activity, demonstrating that its cellular roles stretch beyond just being a ppGpp co-regulator and establishing a more divers, independent role of the factor under different stress and towards different transcription complexes. We show that, in addition to its role during initiation, DksA has many features of an elongation factor: it inhibits both RNA chain extension and RNA shortening by exonucleolytic cleavage or pyrophosphorolysis and increases intrinsic termination in vitro and in vivo. Interestingly, despite the fact that both GreB and DksA target elongation complexes (EC), GreB does not compete with DksA during termination whereas DksA, even when present in several hundredfold molar excess, does not inhibit GreB-mediated cleavage of the nascent RNA in backtracked elongation complexes. Structural modeling suggests that i6, an insertion in the catalytic trigger loop, hinders DksA access into the channel, restricting DksA action to a subset of transcription complexes and preventing competition between the two factors. In support of this hypothesis, we demonstrate that deletion of i6 permits DksA binding to ECs and that the distribution of DksA and i6 in bacterial genomes is strongly concordant. We hypothesize that DksA binds to transcription complexes in which i6 becomes mobile, for example, as a consequence of weakened RNAP interactions with the downstream duplex DNA.
s1.1 is a subdomain of the s70 transcription factor that is essential in vivo and alters transcription initiation in vitro in a promoter-specific manner. Here we report that s1.1 is required for the regulation of the ribosomal promoters by DksA. Our data indicate that deletion of s1.1 changes the properties of RPO. We suggest that the deletion alters the path of isomerization during transcription initiation, thus, preventing the destabilization of RPO by DksA.
Previous work by Blankschien et al., suggested that a conformation change in DksA that may be induced by a substitution at its Asn88 position. In this work we demonstrate that DksA indeed goes through a conformational change at lower pH; however we note that residues at the 88 position do not participate in the observed change. We speculate that the change plays a role in the essential, ppGpp-independent, activity of DksA during acid stress.
DksA proteins studied to date contain a canonical Cys-4 Zn-finger motif thought to be essential for their proper folding and thus activity. Here, we charchterize a novel member of the secondary channel- binding proteins, DksA2 , which can functionally substitute for the canonical DksA in vivo in Escherichia coli and Pseudomonas aeruginosa. We demonstrate that DksA2 shares an almost identical structure and functional characteristics with DksA despite the lack of a Zn finger. dksA2 expression is increased at low Zn concentrations. Our data suggest that DksA2 plays a role in Zn homeostasis and serves as a back-up copy of the canonical Zn-dependent DksA in Zn-poor environments.