A translational riboswitch coordinates nascent transcription–translation coupling

Abstract

Bacterial messenger RNA (mRNA) synthesis by RNA polymerase (RNAP) and first-round translation by the ribosome are often coupled to regulate gene expression, yet how coupling is established and maintained is ill understood. Here, we develop biochemical and single-molecule fluorescence approaches to probe the dynamics of RNAP–ribosome interactions on an mRNA with a translational preQ1-sensing riboswitch in its 5′ untranslated region. Binding of preQ1 leads to the occlusion of the ribosome binding site (RBS), inhibiting translation initiation. We demonstrate that RNAP poised within the mRNA leader region promotes ribosomal 30S subunit binding, antagonizing preQ1-induced RBS occlusion, and that the RNAP–30S bridging transcription factors NusG and RfaH distinctly enhance 30S recruitment and retention, respectively. We further find that, while 30S–mRNA interaction significantly impedes RNAP in the absence of translation, an actively translating ribosome promotes productive transcription. A model emerges wherein mRNA structure and transcription factors coordinate to dynamically modulate the efficiency of transcription–translation coupling.

RfaH Counter-Silences Inhibition of Transcript Elongation by H-NS–StpA Nucleoprotein Filaments in Pathogenic Escherichiacoli

“…Of these 22 genes, 77% had RNAP signal above background (Class II-VI), but all lacked RfaH signal ( Data set S2E ). RfaH binding at these sites may be excluded by NusG bound to coupled transcription–translation complexes ( 69 – 72 ). We conclude that CFT073 encodes 8 bona fide RfaH-regulated loci all bound by H-NS, supporting the RfaH counter-silencing model.…”

Synthesis of O6-alkylated preQ1 derivatives

“…d]pyrimidine also termed prequeuosine base (preQ 1 ) [6,7]. In many bacteria, preQ 1 binds to specific mRNA domains and thereby regulates genes that are required for queuosine biosynthesis [8][9][10][11][12][13][14][15][16]. The molecular mechanism behind is called riboswitching.…”

Creation of Architecturally Minimal Transcriptionally Activating Riboswitches Responsive to Theophylline Reveals an Unconventional Design Strategy

“…However, this strategy has rarely been used to design transcription-activating riboswitches. For riboswitches that regulate translation, the Shine–Dalgarno (SD) sequence is sequestered or freed upon binding of trigger molecules, thereby initiating or blocking translation. , Since the SD sequences consist of few conserved nucleotides, Watson–Crick pairing is usually highly effective in sequestering this region, resulting in blockade of translation so that keeping the OFF-state. Conversely, de novo design of transcription-ON riboswitches requires the construction of large random mutagenesis libraries to screen for the switchable terminators, which ensure that they are changed to antiterminators upon binding to the trigger molecule and remain functional for switching .…”