Role of DNA bubble rewinding in enzymatic transcription termination

Abstract

By using DNA heteroduplexes that inhibit rewinding of the upstream part of the transcription bubble, we show that transcript release in termination by the enzymes Mfd and Rho is facilitated by reannealing of DNA in the upstream region of the transcription bubble, as is also true for termination by intrinsic terminators. We also show that, like Mfd, the Rho termination factor promotes forward translocation of RNA polymerase. These results support termination models in which external forces imposed on nucleic acids induce concerted rewinding of DNA and unwinding of the DNA͞RNA hybrid, possibly accompanied by forward translocation of RNA polymerase, leading to transcription complex dissociation.RNA polymerase ͉ Mfd protein ͉ Rho termination factor T ermination of transcription and the release of RNA polymerase (RNAP) from its templating complex with DNA are essential for providing a boundary for gene expression and removing stalled enzymes that may obstruct gene expression and replication. Three mechanisms are known that cause the otherwise notably stable transcription complex of Escherichia coli RNAP to dissociate. These mechanisms are (i) the intrinsic terminator, consisting of nucleic acid structures that interact with RNAP (1), (ii) the termination factor Rho, an RNAdependent ATPase and RNA ''helicase'' (or, more accurately, RNA translocase) that acts by binding the emerging transcript and (presumably) RNAP (2, 3), and (iii) Mfd, an ATPdependent DNA translocase that acts on RNAP and DNA upstream of the transcription bubble (4-6) ( Fig. 1). [The replication fork apparatus could contain a fourth mechanism that removes obstructing transcription complexes (7).] Understanding these termination pathways may reveal important aspects of transcription complex stability, as well as the nature of regulation that acts through antitermination.Two classes of models of termination describe how nucleic acids could move relative to the enzyme such that the RNA becomes weakly held and can dissociate, leading to RNAP dissociation. The first class of models (''mechanical models''), illustrated in Fig. 2, proposes that rewinding of the upstream boundary of the transcription bubble is coupled with unwinding of the RNA͞DNA hybrid within the enclosing structure of the enzyme (Fig. 2 B and C). In one mechanical model (Fig. 2B), the enzyme translocates forward without RNA synthesis, retaining protein-nucleic acid contacts of the elongation complex (8, 9); in another (Fig. 2C), the transcription bubble collapses within the channel as the hybrid unwinds, without enzyme translocation. The second class of models (''allosteric'') is less defined but proposes that long-range conformational changes in RNAP induced by some element of the terminator (e.g., the RNA hairpin or activities of the enzymatic terminators) destabilize the enzyme-nucleic acid contacts and lead to complex collapse (10).One apparent similarity among intrinsic and enzymatic terminators favoring mechanical models is that all three involve forces exerted on upstream nucleic a...

Dissociation of halted T7 RNA polymerase elongation complexes proceeds via a forward-translocation mechanism

“…4A are aimed to test these predictions (10). As predicted by the model, those constructs containing a mismatch at the upstream end of the bubble show a large increase in complex lifetime, whereas similar mismatches at the downstream end show no detectable effect.…”

“…3A) or remove the need to melt downstream DNA (down mm in Fig. 3A) (10). Both constructs are based on the control construct described in Fig.…”

“…A run of encoded U's has been proposed both to slow transcription and to weaken the RNA-DNA hybrid. Recently, it has been proposed that, rather than direct dissociation of the nascent transcript from a complex halted at the primary termination site (with or without allosteric assistance), dissociation occurs primarily from forward-translocated states (9)(10)(11). In forwardtranslocated (hypertranslocated) states, the hybrid is shortened, weakening the binding of the RNA to the complex.…”

Polymerase trafficking

“…Mfd is an ATP-dependent DNA translocase that binds to the rear of a halted elongation complex and rewinds the transcription bubble behind the RNAP. 23,24 Depending on whether conditions for forward translocation along the DNA are unfavorable or favorable, Mfd binding results in displacement or elongation of the polymerase, respectively. 25 Mfd also binds to the NER factor UvrA and therefore recruits the DNA repair machinery to the stall site.…”

Transcription Elongation Complex Stability

“…In the study of elongation complex stability in vitro, one can artificially omit one of the substrate nucleoside triphosphates, leading to a halting of elongation at the first occurrence of the omitted nucleotide (11,12). The stability of a halted elongation complex depends on numerous factors, but it has been recently demonstrated that dissociation occurs primarily via a mechanism in which forward translocation of the complex (without incorporating nucleotides into the RNA) leads to a shortening of the RNA-DNA hybrid (11, 13).…”