Transcription Elongation Factor NusA Is a General Antagonist of Rho-dependent Termination in Escherichia coli

Abstract

NusA is an essential protein that binds to RNA polymerase and also to the nascent RNA and influences transcription by inducing pausing and facilitating the process of transcription termination/antitermination. Its participation in Rho-dependent transcription termination has been perceived, but the molecular nature of this involvement is not known. We hypothesized that, because both Rho and NusA are RNA-binding proteins and have the potential to target the same RNA, the latter is likely to influence the global pattern of the Rho-dependent termination. Analyses of the nascent RNA binding properties and consequent effects on the Rho-dependent termination functions of specific NusA-RNA binding domain mutants revealed an existence of Rho-NusA direct competition for the overlapping nut (NusA-binding site) and rut (Rho-binding site) sites on the RNA. This leads to delayed entry of Rho at the rut site that inhibits the latter's RNA release process. High density tiling microarray profiles of these NusA mutants revealed that a significant number of genes, together with transcripts from intergenic regions, are up-regulated. Interestingly, the majority of these genes were also up-regulated when the Rho function was compromised. These results provide strong evidence for the existence of NusA-binding sites in different operons that are also the targets of Rho-dependent terminations. Our data strongly argue in favor of a direct competition between NusA and Rho for the access of specific sites on the nascent transcripts in different parts of the genome. We propose that this competition enables NusA to function as a global antagonist of the Rho function, which is unlike its role as a facilitator of hairpin-dependent termination.The bacterial transcription terminator, Rho, functions as a hexameric RNA-dependent ATPase that is capable of translocating along the RNA. It dislodges the elongating RNA polymerase (RNAP) 4 once it is loaded onto the nascent RNA (1, 2). Its termination function is facilitated upon interaction with the transcription elongation factor, NusG (3-5). Recent genomics studies have revealed that Rho-dependent termination occurs at more than one-third of the operons of a dividing Escherichia coli (6, 7). This mode of transcription termination is involved in many physiological processes, like control of translation (2), riboswitch formation (8), inhibition of unwanted antisense transcription, etc. (7). Rho is capable of loading onto unstructured stretches of RNA (the rut ([R]ho [ut]ilization) sites), and once it is bound to the RNA, it translocates in a processive manner along the 5Јto 3Ј direction, which may induce unwanted termination of transcription. Interestingly, in vivo negative regulation by cellular factors or any control switch of the Rho function has not been documented. In principle, any RNA-binding protein that has overlapping binding sites with the rut sites on the nascent RNA could influence the Rho activity by a direct competition for occupancy of the same sites.NusA, a ubiquitous transcription elo...

RNA-binding proteins in bacteria

“…In Bacillus subtilis, 25% of all termination events are ascribed to NusA, especially at sub-optimal intrinsic terminators 29 . Not only does this protein affect intrinsic termination: E. coli NusA may also globally antagonize Rho function 30 .…”

Ancient Transcription Factors in the News

“…λN remodels the β flap domain, which forms one wall of the RNA exit channel, and the RNA-binding domains of NusA to redirect the nascent RNA away from the RNA exit tunnel where formation of a terminator hairpin, stabilized by NusA in the absence of λN (89), would trigger inactivating changes in RNAP (43, 44). These interactions explain how intrinsic termination is disfavored (90) and why Rho may fail to dissociate λN-TAC (91, 92): Rho has to track along the nascent RNA to trigger termination and could thus be sterically blocked from accessing RNAP by NusA domains. The λN-TAC structure also reveals that NusE interacts with L1 and L2 loops of the NusG-CTD, i.e., the same region that binds to Rho (32), thereby preventing NusG activation of Rho through direct exclusion.…”

“…However, while the evidence for S4 contribution to antitermination is solid, it is unclear whether S4 is principally responsible for the potent antitermination activity of the rrn -TAC. Secondary RNA structures, stabilized by NusA (or S4), could hinder Rho access (91, 96), but Rho is able to terminate synthesis of highly structured tRNAs (99). Antipausing properties of NusG unmasked by NusE contacts to the NusG-CTD (28) are unlikely to explain Rho inhibition because a much more potent antipausing activity of RfaH (15) is largely dispensable for its anti-Rho effects (12), which are instead due to RfaH activation of translation (27).…”

Mechanism of Action of Bacterial Transcription Terminator Rho

“…Another mode of action of NusA could be that the NusA binding region on RNA may overlap with that of the Rho binding region. A recent study described that NusA mutants in SKK domain with enhanced RNA binding affinity were able to inhibit Rho dependent termination at the λt R1 (having an overlapping nut site) more efficiently by competing with the Rho loading step (Qayyum et al, 2016). It is expected that there exist overlapping NusA and Rho binding sites on many different mRNAs.…”