Coupling of Downstream RNA Polymerase–Promoter Interactions with Formation of Catalytically Competent Transcription Initiation Complex

Abstract

Bacterial RNA polymerase (RNAP) makes extensive contacts with duplex DNA downstream of the transcription bubble in initiation and elongation complexes. We investigated the role of downstream interactions in formation of catalytically competent transcription initiation complex by measuring initiation activity of stable RNAP complexes with model promoter DNA fragments whose downstream ends extend from +3 to +21 relative to the transcription start site at +1. We found that DNA downstream of position +6 does not play a significant role in transcription initiation when RNAP-promoter interactions upstream of the transcription start site are strong and promoter melting region is AT-rich. Further shortening of downstream DNA dramatically reduces efficiency of transcription initiation. The boundary of minimal downstream DNA duplex needed for efficient transcription initiation shifted further away from the catalytic center upon increasing the GC content of promoter melting region or in the presence of bacterial stringent response regulators DksA and ppGpp. These results indicate that the strength of RNAP-downstream DNA interactions has to reach a certain threshold to retain the catalytically competent conformation of the initiation complex and that establishment of contacts between RNAP and downstream DNA can be coupled with promoter melting. The data further suggest that RNAP interactions with DNA immediately downstream of the transcription bubble are particularly important for initiation of transcription. We hypothesize that these active center-proximal contacts stabilize the DNA template strand in the active center cleft and/or position the RNAP clamp domain to allow RNA synthesis.

TraR directly regulates transcription initiation by mimicking the combined effects of the global regulators DksA and ppGpp

“…For DksA, this conformational change (in the presence of ppGpp) was proposed to involve allosteric effects of an interaction of the C-terminal helix with the SI1 region the β-subunit (14). These interactions could impact interactions of the DksA coiled-coil tip aspartate residues with the trigger loop/bridge helix of RNAP, which could in turn allosterically affect promoter DNA interactions in the main channel (23,34). Alternatively, β-subunit-SI1 interactions could affect promoter binding by altering the downstream DNA binding interface in β (34,35).…”

“…These interactions could impact interactions of the DksA coiled-coil tip aspartate residues with the trigger loop/bridge helix of RNAP, which could in turn allosterically affect promoter DNA interactions in the main channel (23,34). Alternatively, β-subunit-SI1 interactions could affect promoter binding by altering the downstream DNA binding interface in β (34,35). Interactions of TraR with the trigger loop/active site region and/or β SI1 would thus mimic the DksA TraR * * * * * * * * ** * * * * ** * * * * allosteric changes caused by ppGpp binding to site 2 in the RNAP-DksA complex.…”

RNA polymerase molecular beacon as tool for studies of RNA polymerase–promoter interactions

“…DksA/ppGpp inhibit transcription by destabilizing short-lived RP o complexes formed on sensitive promoters [43,51]. Beacon assay measurements revealed that DksA/ppGpp decrease RNAP affinity for downstream fork junction probes by 16-fold, while slightly improving RNAP binding to an upstream fork junction and exerting no effect on RNAP binding to oligo probes similar to those shown in Figure 3 [16]. These experiments indicate that DksA and ppGpp considerably weaken the RNAP interaction with the downstream DNA duplex.…”

“…These experiments indicate that DksA and ppGpp considerably weaken the RNAP interaction with the downstream DNA duplex. Together with biochemical data, the results suggested that weakening of downstream RNAP − promoter contacts by DksA/ppGpp may be the cause of specific inhibition of transcription initiation from promoters on which the RP o formation is relatively energetically unfavorable [16]. …”

“…RNAP also makes extensive sequence-nonspecific contacts with promoter DNA. While these contacts are not responsible for promoter recognition, they contribute to stabilization of transcription complexes and may facilitate promoter DNA melting [1, 13–16]. Thus, the rate of formation and stability of transcription initiation complexes and, ultimately, the level of transcription is determined by the numerous partial interactions between distinct structural elements of RNAP and segments of promoter DNA.…”