RNA Polymerase Pausing Regulates Translation Initiation by Providing Additional Time for TRAP-RNA Interaction  Alexander V. Yakhnin, Helen Yakhnin, Paul Babitzke  Molecular Cell  Volume 24, Issue 4, Pages 547-557 (November 2006) DOI: 10.1016/j.molcel.2006.09.018 Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 1 Models of B. subtilis trp Operon Transcription Attenuation and trpE Translation Control Transcription attenuation model (top). During transcription, RNAP pauses after synthesis of U107. Under tryptophan-limiting conditions, TRAP is not activated and does not bind to trp leader RNA. RNAP eventually overcomes the pause and resumes transcription. In this case, formation of the antiterminator structure prevents formation of the terminator hairpin, resulting in transcription readthrough. Under tryptophan-excess conditions, tryptophan-activated TRAP binds to the (G/U)AG repeats, thereby releasing paused RNAP and simultaneously preventing formation of the antiterminator structure. As a consequence, formation of the terminator hairpin causes transcription to terminate at G140 or U141. Because termination is never 100% efficient, a fraction of RNAP molecules will not terminate in the leader despite the presence of bound TRAP. trpE translation control model (bottom). During transcription of trp operon readthrough transcripts, RNAP pauses after synthesis of U144. Under tryptophan-limiting conditions, TRAP is unable to bind to the nascent trp leader transcript. RNAP eventually overcomes the pause and resumes transcription. In this case, the RNA adopts a structure such that the trpE SD sequence is single stranded and available for ribosome binding. Under tryptophan-excess conditions TRAP can bind to the transcript paused at U144. RNAP eventually overcomes the pause and resumes transcription, which leads to formation of the trpE SD sequestering hairpin and inhibition of translation. Molecular Cell 2006 24, 547-557DOI: (10.1016/j.molcel.2006.09.018) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 2 The Identity of the 3′ Nucleotide Is Critical for B. subtilis RNAP Pausing In Vitro Single-round in vitro transcription reactions were performed with WT and mutant trp leaders in the absence or presence of NusA. Gel slices of the region surrounding residues 144 (top) and 107 (bottom) are shown. Transcription reactions were stopped at the times indicated above each lane (seconds). Reactions corresponding to “chase” were extended for an additional 10 min in the presence of 500 μM of each NTP. Lanes marked with M correspond to an A (top) or G (bottom) RNA sequencing ladder. Molecular Cell 2006 24, 547-557DOI: (10.1016/j.molcel.2006.09.018) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 3 In Vitro Permanganate Footprints of WT and Mutant trp Leader Transcription Bubbles (A) Single-round in vitro transcription reactions were performed with WT and mutant DNA templates in the presence of 1 μM NusA. KMnO4 was added at the times indicated above each lane (seconds). Reactions corresponding to chase (Ch) were extended for an additional 5 min in the presence of 500 μM of each NTP before the addition of KMnO4. Positions of selected T residues are shown. (B) Reactions were carried out as for (A) except that 1 μM GreA was added where indicated. Molecular Cell 2006 24, 547-557DOI: (10.1016/j.molcel.2006.09.018) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 4 In Vivo Permanganate Footprints of WT and Mutant trp Leader Transcription Bubbles (A) Primer extension reactions were carried out on WT or mutant plasmid DNA purified from KMnO4-treated WT (mtrB+) or TRAP-deficient (ΔmtrB) B. subtilis cells. trp leader mutations and a mock-treated DNA control (−KMnO4) are indicated above each lane. Positions of T107, T142, and T144 are marked. (B) RNAP pause half-life determination in vivo. Cells were harvested at the times indicated above each lane (min) after rifampicin addition. Primer extension reactions were carried out on WT plasmid DNA purified from KMnO4-treated ΔmtrB cells. (C) Primer extension reactions were carried out on WT or mutant plasmid DNA purified from KMnO4-treated ΔmtrB cells. Cells also contained the ΔgreA allele where indicated. Molecular Cell 2006 24, 547-557DOI: (10.1016/j.molcel.2006.09.018) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 5 S1-Nuclease Mapping of the 3′ End of the U144 Paused RNA Total cellular RNA purified from ΔmtrB cells was hybridized with a labeled DNA probe and subsequently treated with S1 nuclease (lane S1). A control reaction with probe only (no RNA) is shown. Selected residues are marked. Lanes corresponding to Maxim and Gilbert sequencing reactions of the probe used in this analysis are indicated. Molecular Cell 2006 24, 547-557DOI: (10.1016/j.molcel.2006.09.018) Copyright © 2006 Elsevier Inc. Terms and Conditions