A Critical Role of Downstream RNA Polymerase-Promoter Interactions in the Formation of Initiation Complex

Abstract

Formation of the transcription-competent open promoter complex (RP o ) by DNA-dependent RNA polymerase (RNAP) 3 is a critical checkpoint on the pathway of gene expression. In bacteria, the transition from initial recognition of the promoter by RNAP to RP o proceeds through a series of short-lived intermediate complexes (1-3). In RP o , the DNA duplex is disrupted over a stretch of 12-15 base pairs, which leads to the formation of the transcription bubble and makes the transcription start point (position ϩ1) accessible to the RNAP catalytic center (4, 5). An important source of energy driving this strand separation process is the interaction of the RNAP 70 subunit region 2 with the non-template strand of the Ϫ10 promoter element at the upstream edge of the transcription bubble (6 -9). The interaction with the Ϫ10 element adenine at position Ϫ11 of the nontemplate strand is of special importance for nucleation of promoter melting (6, 10). RNAP interaction with the template segment of the transcription bubble was estimated to be considerably weaker than that with the non-template segment (8). Although formation of intermediates on the pathway to RP o has been characterized kinetically in many studies (1-3, 11, 12), it is still unclear which energetically favorable RNAP-promoter interactions are coupled with downstream propagation of the transcription bubble and determine its final boundary position at ϩ2/ϩ3.The crystal structures of prokaryotic RNAP indicate that establishment of RNAP interactions with the downstream duplex upon RP o formation must introduce a sharp kink in DNA, which may facilitate melting and stabilize the open complex (13,14). Indeed, RNAP mutations that change RNAP contacts with downstream DNA reduce the longevity of open complexes formed at certain promoters (15, 16). On highly supercoiled DNA, the downstream interactions may not be obligatory, as an RNAP subassembly containing an N-terminal fragment of the Escherichia coli RNAP ␤Ј subunit (lacking ␤Ј amino acids involved in contacts with downstream DNA) and a fragment of 70 subunit was reported to recognize and melt an extended Ϫ10 consensus promoter (17). However, melting observed in these experiments might not necessarily mirror steps in RP o formation by RNAP (3, 18). Downstream RNAPpromoter contacts are targeted by low molecular weight inhibitors and protein repressors that affect transcription initiation (16, 19 -21).Unraveling the role of downstream RNAP-promoter interactions in open complex formation and regulation of transcription initiation is hindered by the lack of experimental tools to directly measure their strength and specificity. Upon formation of the open promoter complex, two DNA fork junction structures are created around positions Ϫ12 and ϩ2. Studies of RNAP interactions with model DNA fragments mimicking the upstream fork junction provided important structural (22) and functional insights into the processes of promoter recognition and melting (8,9,(23)(24)(25)(26). Here, we introduce downstream fork junction fragments as ...

Insights into the Mechanism of Initial Transcription in Escherichia coli RNA Polymerase

“…2C the results with a construct, pss(ϩ4), analogous to a construct studied with T7 RNA polymerase, in which the nontemplate strand is removed completely downstream of position ϩ4. Although T7 RNA polymerase initiates well with a downstream single-stranded template (36,37), E. coli RNA polymerase has been shown to require duplex DNA downstream for proper initiation (38). The results of Fig.…”

“…2E, complexes that yield relatively unstable halted complexes at ϩ8 (extensive turnover in ϪGreB measurements) also yield extensive cleavage products in the presence of GreB. Partially single-stranded controls, pss(Ϫ1) and pss(ϩ4) show little accumulation of GreB cleavage products, most likely because they are expected (and observed) to initiate very poorly (38).…”

“…Holoenzyme Abortively Cycles in the Absence of ScrunchingTo probe more directly whether pushing of the 3.2 linker on the growing RNA-DNA hybrid facilitates abortive release of initially transcribing RNA in the native enzyme, we characterized transcription from fork junction DNA constructs, which allow initiation in the complete absence of (38,47). In these constructs, the nontemplate strand of the DNA extends from upstream position Ϫ12 to downstream position ϩ35, whereas the template strand extends from only Ϫ4 upstream to ϩ35 downstream.…”

A Matter of Location: Influence of G-Quadruplexes on Escherichia coli Gene Expression

“…A formed quadruplex structure might simplify strand separation supporting the helicase activity of RNA polymerase. In the region downstream of the À10 region, the polymerase actively separates the double helix (Mekler et al, 2011;Roberts and Roberts, 1996). This might explain why the insertion of a quadruplex in the promoter results in transcriptional repression, whereas location downstream of the promoter increases gene expression.…”

“…The s 70 factor of the E. coli RNA polymerase core enzyme is responsible for promoter recognition, binding the À10 and À35 regions of the double-stranded promoter and unwinding dsDNA at the À10 region, followed by binding to the sense strand. An initial transcript of $10 nt causes the release of the s 70 factor from the core RNA polymerase, which leaves the promoter and enters the elongation phase by moving along the antisense strand (Mekler et al, 2011;Roberts and Roberts, 1996). Promoter recognition can be strongly influenced by the nucleotide composition of the adjacent 5 0 -UTR.…”