Transcription initiation factor DksA has diverse effects on RNA chain elongation

Abstract

Bacterial transcription factors DksA and GreB belong to a family of coiled-coil proteins that bind within the secondarychannel of RNA polymerase (RNAP). These proteins display structural homology but play different regulatory roles. DksA disrupts RNAP interactions with promoter DNA and inhibits formation of initiation complexes, sensitizing rRNA synthesis to changes in concentrations of ppGpp and NTPs. Gre proteins remodel the RNAP active site and facilitate cleavage of the nascent RNA in elongation complexes. However, DksA and GreB were shown to have overlapping effects during initiation, and in vivo studies suggested that DksA may also function at post-initiation steps. Here we show that 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. However, DksA has no effect on Rho- or Mfd-mediated RNA release or nascent RNA cleavage in backtracked complexes, the regulatory target of Gre factors. Our results reveal that DksA effects on elongating RNAP are very different from those of GreB, suggesting that these regulators recognize distinct states of the transcription complex.

Distinct pathways of RNA polymerase regulation by a phage-encoded factor

“…The addition of ppGpp increased the termination efficiency by X. oryzae RNAP (from 52.7 to 62.6%) (Fig. 6C and Table S1), which is similar to its effects on termination by E. coli RNAP (31,32). Furthermore, the p7 titration experiments demonstrated that ppGpp decreased the antitermination efficiency at most p7 concentrations and decreased the apparent affinity of p7 to the TEC (K d ∼265 nM in comparison with ∼130 nM in the absence of ppGpp).…”

DksA regulates RNA polymerase in Escherichia coli through a network of interactions in the secondary channel that includes Sequence Insertion 1

“…DksA is known to alter the elongation properties of RNAP (3,20,30), and we note that DksA statically bound to RNAP as found in our model would preclude elongation by preventing folding of the β' TL. Our experimental efforts focused on the effects of DksA during initiation, but the position of the DksA coiled-coil in the channel may be dynamic and vary with RNAP conformation, the stage of transcription cycle, and the presence of additional factors such as ppGpp.…”

“…This could alter the conformation of two neighboring mobile elements of β, fork loop-1 and fork loop-2, destabilizing the intermediate on the pathway to open complex formation. Alternatively, the β' trigger loop (β' TL) has been previously demonstrated to be essential for sensitivity to DksA (7,20). Our model predicts a steric clash between a folded β' TL and the coiled-coil of DksA, and an alternative mechanistic role of both DksA-D74 and DksA-91 could be to lock the coiled-coil in the appropriate orientation to mediate this interaction.…”

“…The model predicted that the Zn 2+ -binding domain of DksA (G112-K139) contacted the tip of the β' rim helices (β'670-674) (Fig. 3B), a feature of RNAP that is known to be critical for DksA binding (12,16,20). Multiple mutations in both sides of the proposed interface identify key residues that contribute to DksA-binding affinity.…”

Regulation of transcriptional pausing through the secondary channel of RNA polymerase

“…coli Gre factors and DksA were also shown to target different types of the TEC, thus avoiding direct competition between these factors during transcription elongation (38). Although the exact TEC state targeted by DksA remains to be identified, both DksA and Gre ultimately suppress formation of backtracked complexes by E. coli RNAP and decrease the transcription/replication conflicts (5,39).…”