Allosteric control of the RNA polymerase by the elongation factor RfaH

Abstract

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 Å 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 λQ) utilize common strategies during both recruitment and anti-pausing modification of the transcription complex.

Allosteric control of catalysis by the F loop of RNA polymerase

“…Indeed, previous observations pinpointed another insertion, the 188-residue-long SI3 segment in the TL in E. coli RNAP (see Fig. 5 for its position), to play an essential role in catalysis, and its deletion had complex effects on transcription elongation, pausing, and RNA cleavage (3,13). Thus, further structural analysis of the conformational dynamics of the tripartite F-loop/TL/BH unit will provide important insights into the mechanisms of catalysis by multisubunit RNAP.…”

“…Its effect on catalysis may be explained by altered contacts between the BH and the F loop which, in its turn, interacts with the TL. Substitutions that affect the rate of RNA synthesis by E. coli RNAP were mapped to adjacent positions in the BH (13,31). One of these substitutions, F773V (corresponds to Taq F1071; Fig.…”

“…10) or in the BH (F773V in E. coli RNAP; ref. 13), and many substitutions in Methanocaldococcus jannaschii (14), lead to moderate increase in the rate of RNA synthesis.…”

The elongation factor RfaH and the initiation factor σ bind to the same site on the transcription elongation complex

“…2 and many others the isolated N-domain reduces pausing (e.g., at the hisP site) on average by 3-fold. To exclude the indirect model, we used a ''fast'' RNAP variant (␤Ј ⌬943-1130 ) that is defective in both pausing and response to RfaH yet retains the ability to bind to RfaH (14). The mutant enzyme paused at P as efficiently as the WT RNAP (Fig.…”

Transcriptional pausing coordinates folding of the aptamer domain and the expression platform of a riboswitch

“…Both enzymes fail to efficiently pause at strong regulatory sites (e.g., hairpin-dependent hisP and backtracked opsP sites) and bypass numerous weak pause sites more readily (29). In btuB riboswitch transcription, both mutant enzymes show significantly different pausing behavior at sites A-C as compared to the wild-type RNAP (Fig.…”