Transcription Factor Rho Does Not Require a Free End to Act as an RNA-DNA Helicase on an RNA

Abstract

Escherichia coli Rho factor is a ring-shaped, homohexameric protein that terminates synthesis of RNA through interactions with the nascent RNA transcript. Because its mechanism of action may involve translocation of the RNA transcript through the hole in its ring structure, its action could depend on the availability of a free 5 terminus. To determine whether Rho's activity is 5-end-dependent, its ability to bind to and function on a circular derivative of cro mRNA was investigated. The circular derivative was made in vitro by action of RNA ligase on a derivative of cro RNA containing an extra 10-nucleotide sequence near the 5-end that was complementary to a sequence located near the 3-end. Rho bound nearly as tightly to the circular derivative RNA as to the standard cro transcript. Rho was also able to readily dissociate a DNA oligonucleotide from its helical complex with the circular RNA in an ATP-dependent reaction. Thus, the action of Rho on a transcript does not depend on the availability of a free 5 terminus.Transcription termination factor Rho is an RNA-binding protein that couples the energy derived from ATP hydrolysis to actions that dissociate a RNA transcript from its biosynthetic complex with DNA and RNA polymerase (1, 2). Rho also can dissociate a RNA molecule bound to DNA through a short hybrid helix in a reaction that is dependent on ATP hydrolysis and the presence of an attachment site for Rho on the RNA on the 5Ј side of the hybrid helix (3-5). This RNA-DNA helicase activity may mimic the process of removal of the nascent RNA strand from the transcription elongation complex.Rho factor is believed to function in vivo as a hexamer consisting of six polypeptide subunits arranged in a ring-shaped structure (6, 7). Burgess and Richardson (8) have recently presented a model for the Rho-RNA complex in which a segment of RNA, called a rut site (Rho utilization site), binds to a cleft comprised of the N-terminal RNA-binding domains of the six individual subunits located at one end of the hexameric structure (the crown) (9 -11). RNA sequence 3Ј of the rut site then passes into the hole located at the center of the hexamer. Evidence for passage of a single-stranded nucleic acid substrate through a ring-shaped hexameric structure has been observed for other hexameric helicases, such as DnaB and the T7 gene 4 product (12, 13).The process by which Rho binds to and captures the 3Ј-end of an RNA in the center of the hexamer is not known. Gan and Richardson (14) have recently presented data indicating that Rho, in vitro, could form hexamers by partial assembly of subunits on an RNA. Another mechanism could have the RNA enter into the center of the hexameric structure through an opening or notch in the ring (7). A third mechanism may involve Rho threading onto the nascent transcript from the free 5Ј-end of the RNA. To test the model in which Rho requires a free end of RNA to thread onto in order to function as a terminator, we investigated Rho's ability to utilize its ATP-dependent helicase activity on an RNA lac...

Influence of Substrate Composition on the Helicase Activity of Transcription Termination Factor Rho: Reduced Processivity of Rho Hexamers during Unwinding of RNA–DNA Hybrid Regions

“…At various times, aliquots were removed from the reaction mixture and analyzed by PAGE to determine the amount of oligonucleotides released from the RNA transcripts (the loading buffer and gels contained 0.5% SDS to disrupt Rho-RNA complexes; see Materials and Methods). 33,38 When either Rho, Mg 2C , or ATP was absent from the reaction mixture, the RNA-DNA helices were stable for extended periods of time (t 1/2 Othree hours; Figure 2(A), and data not shown). In the presence of all three components, however, time-dependent release of the 32 P-labeled oligonucleotides was observed with a variety of R24, R25, or R26-derived substrates (such as R26-D4 in Figure 2(A)).…”

In Vivo Dynamics of Intracistronic Transcriptional Polarity

“…Rho binds directly to the mRNA at the rut site without scanning the RNA from its 5′ end. 60 The minimal window for binding is therefore determined by the physical footprint of Rho and its binding rate to the RNA. On the one hand, however, Rho's footprint on RNA is only 60-80 nt (see above), and it efficiently terminates ribosome-free transcripts of less than 100 nt.…”

RNA remodeling by hexameric RNA helicases

“…11 Threading of a free RNA end through the Rho ring was also ruled out by experiments in which the ec Rho helicase efficiently dissociated a DNA strand from a circular RNA. 34 Moreover, both electron micrographs [35][36][37][38] and crystal structures of ec Rho 39,40 provide evidence for split-open conformations of the hexamer. Transient openings of the Rho ring therefore provide the most likely means for entry of the RNA chain into the central channel during the early formation (and activation) of the enzyme-substrate complex.…”

“…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).…”