Ligand-induced and small molecule control of substrate loading in a hexameric helicase

Abstract

Processive, ring-shaped protein and nucleic acid protein translocases control essential biochemical processes throughout biology, and are considered high-prospect therapeutic targets. The E. coli Rho factor is an exemplar hexameric RNA translocase that terminates transcription in bacteria. Like many ring-shaped motor proteins, Rho activity is modulated by a variety of poorly understood mechanisms, including small molecule therapeutics, protein-protein interactions, and the sequence of its translocation substrate. Here, we establish the mechanism of action of two Rho effectors, the antibiotic bicyclomycin and nucleic acids that bind to Rho's 'primary' mRNA recruitment site. Using SAXS and a novel reporter assay to monitor the ability of Rho to switch between open-ring (RNA loading) and closed-ring (RNA translocation) states, bicyclomycin is found to be a direct antagonist of ring closure. Reciprocally, the binding of nucleic acids to its N-terminal RNA recruitment domains is shown to promote the formation of a closed-ring Rho state, with increasing primary site occupancy providing additive stimulatory effects. This study establishes bicyclomycin as a conformational inhibitor of Rho ring dynamics, highlighting the utility of developing assays that read out protein conformation as a prospective screening tool for ring-ATPase inhibitors. Our findings further show that the RNA sequence specificity used for guiding Rhodependent termination derives in part from an intrinsic ability of the motor to couple the recognition of pyrimidine patterns in nascent transcripts to RNA loading and activity.

Molecular mechanisms of substrate-controlled ring dynamics and substepping in a nucleic acid-dependent hexameric motor

“…Together, these data confirm that Rho forms an open lock-washer in solution and further demonstrate that both RNA and nucleotide are required to promote ring closure (5). This RNA-and ATP-dependent conformational change in Rho may correspond to the rate-limiting step previously observed in stopped-flow kinetics measurements of ATP hydrolysis (46), a concept supported by the marked acceleration of both ATPase and ring closure rates in response to primary site occupancy (38,47). , in reference to the RNA sequence and space group in which the protein crystallized) (48), we set out to find a new crystal packing arrangement that could be obtained in the presence of different RNA and nucleotide conditions, but that still possessed at least one complete Rho hexamer per asymmetric unit (so as to image the particle in the absence of crystal symmetry influences).…”

“…The differences in secondary-site RNA binding seen here between poly-U and poly-A bound forms of Rho (along with the preferential ring closure capability of poly-U over poly-A described in the accompanying study (47) indicate that sequence discrimination in the helicase may in part arise from interrogation of RNA sequence at the point where the nucleic acid threads into the ring. In particular, the 3′ kink seen in the new Rho PolyU-P2 1 structure allows a ribose 2′-OH and two pyrimidine bases to interact with an invariant lysine, PolyU-P21 also reveals two additional 3′ bases, but in a pseudo A-form helical arrangement, rather than the kinked conformation.…”

Regulatory interplay between small RNAs and transcription termination factor Rho

“…The CC/UC repeats that make up the Rut site are highlighted in yellow. Brackets denote 11-12 nt spacing between the repeats, consistent with the binding of four adjacent Rho subunits (which could be sufficient for productive Rho-Rut interaction [13,22]). Note that the sequence from E. coli contains two alternative sets of appropriately spaced repeats (Blue and Purple brackets).…”

“…Most of the current knowledge on the structure and function of Rho comes from work with the E. coli protein. Rho is an RNA-binding homohexameric protein that can adopt alternative ring-shaped conformations, one in which ring is open and slightly distorted (i.e., ends slightly offset), the other with the ring closed [13][14][15]. The hexamer is thought to initially interact with the RNA in the open conformation.…”

Tuning the sequence specificity of a transcription terminator

“…The mechanistic basis for this stimulation had remained unclear until the relatively recent development of a biochemical assay that allows for the tracking of Rho ring state (i.e., open vs. closed) in solution. Using this assay, it was discovered that RNAs capable of tightly binding to Rho's primary site (i.e., pyrimidine-rich sequences) markedly potentiate Rho ring closure (Lawson et al, 2016). This observation indicates that Rho's sequence specificity as a transcription termination factor arises in part from RNA sequence-dependent conformational control of the helicase (Figure 1, top left).…”