Structural basis for the function of SuhB as a transcription factor in ribosomal RNA synthesis.

Yong-Heng Huang,Nelly Said,Markus C Wahl,Bernhard Loll

doi:10.1093/nar/gkz290

Yong-Heng Huang, Nelly Said + Show 2 more

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https://doi.org/10.1093/nar/gkz290

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Abstract

Ribosomal RNA synthesis in Escherichia coli involves a transcription complex, in which RNA polymerase is modified by a signal element on the transcript, Nus factors A, B, E and G, ribosomal protein S4 and inositol mono-phosphatase SuhB. This complex is resistant to ρ-dependent termination and facilitates ribosomal RNA folding, maturation and subunit assembly. The functional contributions of SuhB and their structural bases are presently unclear. We show that SuhB directly binds the RNA signal element and the C-terminal AR2 domain of NusA, and we delineate the atomic basis of the latter interaction by macromolecular crystallography. SuhB recruitment to a ribosomal RNA transcription complex depends on the RNA signal element but not on the NusA AR2 domain. SuhB in turn is required for stable integration of the NusB/E dimer into the complex. In vitro transcription assays revealed that SuhB is crucial for delaying or suppressing ρ-dependent termination, that SuhB also can reduce intrinsic termination, and that SuhB-AR2 contacts contribute to these effects. Together, our results reveal functions of SuhB during ribosomal RNA synthesis and delineate some of the underlying molecular interactions.

Highlights

Transcription in bacteria is terminated predominantly via two mechanisms [1]
Intrinsic termination can be enhanced by transcription factor N-utilization substance (Nus) A that binds RNA polymerase (RNAP) and stabilizes RNA hairpins in the exit tunnel [3,4,5,6]. ␳ -dependent termination can be increased by NusG that binds RNAP via its N-terminal domain (NTD) and contacts ␳ via its C-terminal domain (CTD), thereby facilitating clamp-down of ␳ on RNA at sub-optimal rut sites [7]
The structural details underlying formation of a transcription complex that is specialized for rRNA synthesis in E. coli, the rRNA transcription antitermination complex (rrnTAC) that involves RNAP, a nut-like site on the transcript, NusA, B, E and G, r-protein S4 and SuhB, are presently not known

Summary

Introduction

Transcription in bacteria is terminated predominantly via two mechanisms [1]. Intrinsic termination depends on a stable RNA hairpin followed by a sequence rich in uridines; the hairpin invades the RNA exit tunnel of RNA polymerase (RNAP), while the U-rich stretch forms a weak DNA:RNA hybrid, facilitating termination. In ␳ -dependent termination, the hexameric RNA-dependent NTPase, ␳ , engages the nascent transcript at C-rich sequences, so-called ␳ utilization (rut) sites, uses its NTP-dependent RNA translocase activity to track down RNAP and, upon encounter, leads to termination. Both modes of termination can be supported or suppressed by transcription factors [2]. Intrinsic termination can be enhanced by transcription factor N-utilization substance (Nus) A that binds RNAP and stabilizes RNA hairpins in the exit tunnel [3,4,5,6]. NusG can counteract both modes of termination by enhancing RNAP processivity [8,9], while NusA can inhibit ␳ -dependent termination by competing for rut sites [10]

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