Noncanonical Interactions in the Management of RNA Structural Blocks by the Transcription Termination Rho Helicase

Abstract

To trigger transcription termination, the ring-shaped RNA-DNA helicase Rho from Escherichia coli chases the RNA polymerase along the nascent transcript, starting from a single-stranded C-rich Rut (Rho utilization) loading site. In some instances, a small hairpin structure divides harmlessly the C-rich loading region into two smaller Rut subsites, best exemplified by the tR1 terminator from phage lambda. Here, we show that the Rho helicase can also elude a RNA structural block located far downstream from the single-stranded C-rich region but upstream from a reporter RNA-DNA hybrid. In this process, Rho hexamers do not melt the intervening RNA motif but require single-stranded RNA segments on both of its sides. The reaction is also favored by physiological glutamate ions and can implicate Rho primary recognition of 5'-YC dimers (as for Rut binding) significantly upstream (>70 nucleotides) from the intervening motif. Surprisingly, we also found that primary interactions of Rho with 2'-hydroxyl groups located upstream from the intervening RNA structure serve to elude the motif. This demonstrates that the preference of Rho for RNA residues is not limited to the secondary interaction site that mediates ATPase-fuelled mechanochemistry within the hexamer central channel. These features could be part of an energy-effective mechanism in which Brownian exploration of the conformation of the Rho-substrate complex and accommodation of downstream secondary structures within a composite primary interaction site replace ATP-dependent translocation of the Rho enzyme along corresponding structured portions of the RNA chain.

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

“…It is well established that the sequence of the RNA itself has a pronounced impact on whether a transcript will be acted upon by Rho (12,[27][28][29]. Binding of pyrimidine-rich sequences to the N-terminal RNA-binding domains of Rho is a particularly well-known accelerant of Rho's ATPase activity (30), with presteady-state ATPase assays showing that the formation of a catalytically competent Rho ring is governed by a rate-limiting RNA-and ATP-dependent conformational change (8).…”

Expression of Bacterial Rho Factor in Yeast Identifies New Factors Involved in the Functional Interplay between Transcription and mRNP Biogenesis

“…The mechanism by which Rho induces transcription termination involves a primary interaction of the protein with a naked portion of the nascent transcript, followed by ATPdriven translocation and the disruption of the transcription elongation complex at downstream sites. Previous in vitro and in vivo studies in our laboratory have shown that a well-defined Rho-dependent terminator is not an absolute prerequisite for Rho activity; any RNA sequence with adequately spaced C residues or certain RNA structures such as hairpins can serve as efficient loading sites for Rho (18,58). Also, Rho is able to compete with other proteins for binding to a transcript, a process used in prokaryotic gene regulation (66).…”

RNA remodeling by hexameric RNA helicases

“…6A and B). 23 This phenomenon is most likely due to the accommodation of the intervening RNA structure on the PBS face of the Rho ring (Fig. 6A) during the early activation steps (Fig.…”

“…18 Suitable helicase substrates for the two enzymes contain a 'tracking' RNA strand with a single-stranded region upstream from the duplex region. 16,18 For ec Rho, helicase activity is optimal if the single-stranded region is more than ~70 nt-long and contains a natural 16,17,[20][21][22]34 or synthetic 18,19,23 cytosine-rich Rut (Rho Utilization) loading site. A long (>100 nt) single-stranded region also appears necessary for ϕ8 P4 helicase activity (our unpublished observations).…”

“…The transcription termination factor Rho from E. coli ( ec Rho) unwinds RNA and RNA-DNA helices in a NTP-dependent fashion, [16][17][18][19][20][21][22][23] a property that is likely connected to Rho-induced dissociation of transcription elongation complexes (TECs; composed of the DNA template, RNA polymerase and transcript; Fig. 2B) at specific loci of bacterial genomes, termed Rho-dependent terminators.…”