Allosteric control of the RNA polymerase by the elongation factor RfaH

Vladimir Svetlov 1 Georgiy A. Belogurov 1 Elena Shabrova 1 Dmitry G. Vassylyev 0 Irina Artsimovitch 1 0 Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham , 720 20th Street South, Birmingham, AL 35294, USA 1 Department of Microbiology, The Ohio State University , 484 West 12th Avenue, Columbus, OH 43210 Efficient transcription of long polycistronic operons in bacteria frequently relies on accessory proteins but their molecular mechanisms remain obscure. RfaH is a cellular elongation factor that acts as a polarity suppressor by increasing RNA polymerase (RNAP) processivity. In this work, we provide evidence that RfaH acts by reducing transcriptional pausing at certain positions rather than by accelerating RNAP at all sites. We show that 'fast' RNAP variants are characterized by pause-free RNA chain elongation and are resistant to RfaH action. Similarly, the wild-type RNAP is insensitive to RfaH in the absence of pauses. In contrast, those enzymes that may be prone to falling into a paused state are hypersensitive to RfaH. RfaH inhibits pyrophosphorolysis of the nascent RNA and reduces the apparent Michaelis-Menten constant for nucleotides, suggesting that it stabilizes the post-translocated, active RNAP state. Given that the RfaH-binding site is located 75 A away from the RNAP catalytic center, these results strongly indicate that RfaH acts allosterically. We argue that despite the apparent differences in the nucleic acid targets, the time of recruitment and the binding sites on RNAP, unrelated antiterminators (such as RfaH and jQ) utilize common strategies during both recruitment and anti-pausing modification of the transcription complex. - RNAP is an obligatory processive enzyme that must complete synthesis of the entire RNA chain since the transcripts that are released prematurely cannot re-enter transcription cycle. In bacteria, even in the absence of the tightly condensed chromatin, RNAP still encounters many roadblocks that either stall it temporarily or trigger RNA release. DNA-bound proteins, DNA lesions and various nucleic acid signals that induce pausing, arrest and termination (1) can hinder RNAP progression along the template. Even at saturating substrate concentrations in vitro, RNAP is moving in leaps, with its fast movement along the template punctuated by pauses (2). Pausing plays numerous regulatory roles, is an obligatory step in termination pathways, and likely controls the overall rate of RNA chain elongation (3). RNAP is capable of making very long RNA chains (30 000 nt long in bacteria) but its rate is rather modest compared to DNA replicases: in Escherichia coli, elongating RNAP (a complex of a2bb0! subunits) moves at 2090 nt/s (4) whereas the replication fork advances 1000 nt/s (5). This relatively inefficient operation of RNAP does not represent the limit of its catalytic potential since fast substitutions in the b and b0 subunits that significantly increase its overall rate in vitro have been described (610). An attractive explanation rests on an assumption that the relatively slow rate of transcription is necessary for efficient regulation of gene expression where it provides for timely recruitment of, and response to regulatory factors, attenuation control, as well as determines folding pathways of the nascent RNA. Moreover, in bacteria transcription and translation are coupled, imposing additional restrictions on the speed that RNAP can attain without placing the nascent RNA in danger of release by Rho, which terminates the untranslated messages (4). In other words, a catalytically perfect RNAP would leave little room for regulation and likely uncouple transcription and translation, while much slower RNAP would not be nimble enough to keep up with sustaining the RNA pool as it adapts to changing environmental and physiological conditions. Indeed, while different fast and slow viable alleles of RNAP have been isolated, they alter the apparent elongation rate in vitro by less than 3- to 5-fold in each direction (7,8,1012), whereas mutations coding for much faster or slower enzyme variants are lethal (6,9,1315). As substitutions that constitutively change the overall rate of RNA chain elongation appear to have a negative impact on fitness and are being removed by natural selection, the stage is set for transient alteration of RNAP kinetic properties by regulatory proteins. A subset of such factors (known as antiterminators) reduces pausing and termination (in other words, confers a fast phenotype) thereby helping RNAP transcribe long operons. These proteins use different nucleic acid targets during recruitment: lN binds the nascent RNA structure, lQ is recruited to the double-stranded DNA near the promoter, RfaH is recruited to the single-stranded non-template (NT) DNA strand during elongation (1619). The sites on RNAP to which these proteins bind are likely also distinct: we have recently concluded (20) that RfaH binds to the b0-subunit clamp helices (b0 CH), whereas the target sites for l regulators are still unknown but are thought to be quite different (21,22). Yet all antiterminators share the ability to accelerate RNAP, suggesting that they induce similar changes in the transcription elongation complex (TEC). To date, the changes that lead to the antitermination modification of the RNAP have not been characterized in detail, and the molecular mechanism(s) by which elongation factors or substitutions in RNAP make the enzyme faster or slower is not known: they may control nucleotide addition at every template position by affecting the common rate-limiting step (which has not been elucidated for RNAP), or influence the TEC isomerization into off-pathway states at pause and termination sites (23). We have proposed that at a pause site RNAP isomerizes into a state in which nucleotide addition is slowed due to transient changes in the active site architecture (Figure 1), and from which different classes of pause and termination complexes arise (24). We further speculated that substitutions in RNAP may alter its propensity towards the isomerization into the slow state. In a fast RNAP, the productive alignment of the 30 RNA end in the active site, and consequently nucleotide addition, is favored. In contrast, a slow RNAP is more likely to lose the 30end from the active site and enter a paused state, escape from which can be delayed by two orders of magnitude. Antiterminators may act in the same regulatory pathway, switching RNAP into the fast state. Slow, pause-prone enzymes should then be hypersensitive to modification by antiterminators, whereas fast RNAPs should appear resistant to further acceleration. To test this hypothesis, we have determined effects of the E. coli RfaH on RNA chain elongation by enzymes from an expanded panel of fast and slow RNAPs, including many previously uncharacterized kinetic variants. RfaH is recruited to the TEC at specific sites (called ops) and is required for express (...truncated)