Structural basis of the transcription termination factor Rho engagement with transcribing RNA polymerase from Thermus thermophilus

Abstract

Transcription termination is an essential step in transcription by RNA polymerase (RNAP) and crucial for gene regulation. For many bacterial genes, transcription termination is mediated by the adenosine triphosphate–dependent RNA translocase/helicase Rho, which causes RNA/DNA dissociation from the RNAP elongation complex (EC). However, the structural basis of the interplay between Rho and RNAP remains obscure. Here, we report the cryo–electron microscopy structure of the Thermus thermophilus RNAP EC engaged with Rho. The Rho hexamer binds RNAP through the carboxyl-terminal domains, which surround the RNA exit site of RNAP, directing the nascent RNA seamlessly from the RNA exit to its central channel. The β-flap tip at the RNA exit is critical for the Rho-dependent RNA release, and its deletion causes an alternative Rho-RNAP binding mode, which is irrelevant to termination. The Rho binding site overlaps with the binding sites of other macromolecules, such as ribosomes, providing a general basis of gene regulation.

Structural basis of eukaryotic transcription termination by the Rat1 exonuclease complex

“…Rat1 binds at the RNA-exit site of RNAPII to incorporate RNA into its active site. This view is analogous to the manner in which the bacterial termination factor Rho interacts with transcribing RNAP 44,45 . Although Rho is an ATP-dependent RNA helicase/translocase, it binds the similar part of RNAP (the b-flap and zinc-binding domains) to extract nascent RNA.…”

Sm-like protein Rof inhibits transcription termination factor ρ by binding site obstruction and conformational insulation

“…YihE binds the ρ NTD, inhibiting ρ-RNA interactions (20); the mechanism of ρ regulation by Rof has been determined in this study, and molecular details of Hfq-mediated ρ inhibition remain to be solved (16, 19). By directly binding ρ, each of these anti-terminators may interfere with the formation of ρ-dependent termination complexes that assemble in an EC-dependent manner (center left) (31, 32) or RNA-dependent manner (center right) (47, 48).…”

“…In vitro , ρ-dependent termination can proceed via two pathways (45, 46). In the RNA-dependent pathway, ρ binds C-rich, unstructured and unoccupied RNA regions via its PBSes; subsequent binding of a neighboring RNA region at the SBS and ring closure allow ρ to translocate towards elongating RNAP, eventually contact the transcriptional machinery around the RNA exit channel and terminate transcription (47, 48). In the EC-dependent pathway, ρ associates with and accompanies ECs without immediately terminating transcription (49); upon EC pausing, NusA and NusG can cooperate with ρ in inducing conformational changes in RNAP that stall transcription before ρ engages the transcript (31, 32).…”

“…2B). In each case, Rof interacts primarily with one ρ NTD through its N-terminal α-helix (α1, residues 9-23), the loop connecting strands β3 and β4 (residues [46][47][48][49][50], and the very C-terminal region (Fig. 2C).…”

Mycobacterium tuberculosisemploys atypical and different classes of B12switches to control separate operons

“…Intrinsic transcription termination requires the formation of a stable hairpin, which in the context of a U-rich tail leads to dissociation of the elongation complex (13,14). In the Rho-dependent mechanism, the ATP-dependent RNA helicase Rho binds to exposed C-rich sequences on RNA (known as Rho utilisation (RUT) sites), and translocates along the transcript to disrupt the elongation complex further downstream (15)(16)(17). Riboswitch-mediated translational control typically involves ligand-dependent occlusion of the Shine-Dalgarno sequence (SD) by a complementary anti-SD (αSD) sequence, which, in turn, may be sequestered by an antianti-SD (ααSD) sequence (12).…”