Role of RNA Structure in Transcription Attenuation in Bacillus subtilis: The trpEDCFBA Operon as a Model System

Babitzke, Paul; Schaak, Janell E.; Yakhnin, Alexander V.; Bevilacqua, Philip C.

doi:10.1016/s0076-6879(03)71030-1

Cited by 17 publications

(15 citation statements)

References 14 publications

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“…We created several DNA templates that contain mutant trp leaders with substitutions designed to disrupt base pairing in the A/B antiterminator (19) without altering the (G/U)AG repeats of the TRAP binding site. If the sole function of TRAP binding is to prevent formation of the antiterminator structure, then transcription of these mutant templates should terminate constitutively at the C/D attenuator in the absence of TRAP.…”

Section: Resultsmentioning

confidence: 99%

TRAP binding to the Bacillus subtilis trp leader region RNA causes efficient transcription termination at a weak intrinsic terminator

Potter

Merlino

Jacobs

et al. 2010

Nucleic Acids Research

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The Bacillus subtilis trpEDCFBA operon is regulated by a transcription attenuation mechanism controlled by the trp RNA-binding attenuation protein (TRAP). TRAP binds to 11 (G/U)AG repeats in the trp leader transcript and prevents formation of an antiterminator, which allows formation of an intrinsic terminator (attenuator). Previously, formation of the attenuator RNA structure was believed to be solely responsible for signaling RNA polymerase (RNAP) to halt transcription. However, base substitutions that prevent formation of the antiterminator, and thus allow the attenuator structure to form constitutively, do not result in efficient transcription termination. The observation that the attenuator requires the presence of TRAP bound to the nascent RNA to cause efficient transcription termination suggests TRAP has an additional role in causing termination at the attenuator. We show that the trp attenuator is a weak intrinsic terminator due to low GC content of the hairpin stem and interruptions in the U-stretch following the hairpin. We also provide evidence that termination at the trp attenuator requires forward translocation of RNA polymerase and that TRAP binding to the nascent transcript can induce this activity.

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Section: Resultsmentioning

confidence: 99%

TRAP binding to the Bacillus subtilis trp leader region RNA causes efficient transcription termination at a weak intrinsic terminator

Potter

Merlino

Jacobs

et al. 2010

Nucleic Acids Research

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“…Multi‐round in vitro transcription assays followed a published procedure (Babitzke et al ., 2003). DNA templates contained promoters P1 through P5 (P1–P5), P3 through P5 (P3–P5), P4 and P5 (P4–P5) or P5 only.…”

Section: Methodsmentioning

confidence: 99%

Complex regulation of the global regulatory gene csrA: CsrA‐mediated translational repression, transcription from five promoters by Eσ⁷⁰ and Eσ^S, and indirect transcriptional activation by CsrA

Yakhnin

Baker

et al. 2011

Molecular Microbiology

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124

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Summary CsrA of Escherichia coli is an RNA binding protein that globally regulates gene expression by repressing translation and/or altering the stability of target transcripts. Here we explored mechanisms that control csrA expression. Four CsrA binding sites were predicted upstream of the csrA initiation codon, one of which overlapped its Shine-Dalgarno sequence. Results from gel shift, footprint, toeprint and in vitro translation experiments indicate that CsrA binds to these four sites and represses its own translation by directly competing with 30S ribosomal subunit binding. Experiments were also performed to examine transcription of csrA. Primer extension, in vitro transcription and in vivo expression studies identified two σ70-dependent (P2 and P5) and two σS-dependent (P1 and P3) promoters that drive transcription of csrA. Additional primer extension studies identified a fifth csrA promoter (P4). Transcription from P3, which is indirectly activated by CsrA, is primarily responsible for increased csrA expression as cells transition from exponential to stationary phase growth. Taken together, our results indicate that regulation of csrA expression occurs by a variety of mechanisms, including transcription from multiple promoters by two sigma factors, indirect activation of its own transcription, as well as direct repression of its own translation.

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“…Halted transcription elongation complexes were created as described previously (Babitzke et al 2003). Briefly, B. subtilis RNAP was incubated with the template of interest in 1x transcription buffer (40 mM Tris-HCl (pH 8.0), 4 mM MgCl 2 , 0.6 mM EDTA, 4% trehalose, and 30 mM DTT) with 8 mM ATP and UTP, 2 mM GTP, and 10 mCi [a-32 P]UTP (Perkin-Elmer).…”

Section: Single-round In Vitro Transcription Assaymentioning

confidence: 99%

TRAP-5′ stem–loop interaction increases the efficiency of transcription termination in the Bacillus subtilis trpEDCFBA operon leader region

McGraw¹,

Bevilacqua²,

Babitzke³

2007

RNA

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TRAP regulates expression of the Bacillus subtilis trpEDCFBA operon by a transcription attenuation mechanism in which tryptophan-activated TRAP binds to 11 (G/U)AG repeats in the nascent trp leader transcript. Bound TRAP blocks formation of an antiterminator structure and allows formation of an overlapping intrinsic terminator upstream of the trp operon structural genes. A 59 stem-loop (59SL) structure located upstream of the triplet repeat region also interacts with TRAP. TRAP-59SL RNA interaction participates in the transcription attenuation mechanism by preferentially increasing the affinity of TRAP for the nascent trp leader transcript during the early stages of transcription, when only a few triplet repeats have been synthesized. Footprinting assays indicated that the 59SL contacts TRAP through two discrete groups of single-stranded nucleotides that lie in the hairpin loop and in an internal loop. Filter binding and in vivo expression assays of 59SL mutants established that G7, A8, and A9 from the internal loop, and A19 and G20 from the hairpin loop are critical for proper 59SL function. These nucleotides are conserved among certain other 59SL-containing organisms. Single-round transcription results indicated that the 59SL increases the termination efficiency when transcription is fast; however, the influence of the 59SL was lost when transcription was slowed by reducing the ribonucleoside triphosphate concentration. Since there is a limited amount of time for TRAP to bind to the nascent transcript and promote termination, our data suggest that the contribution of TRAP-59SL interaction increases the rate of TRAP binding, which, in turn, increases the efficiency of transcription termination.

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Role of RNA Structure in Transcription Attenuation in Bacillus subtilis: The trpEDCFBA Operon as a Model System

Cited by 17 publications

References 14 publications

TRAP binding to the Bacillus subtilis trp leader region RNA causes efficient transcription termination at a weak intrinsic terminator

TRAP binding to the Bacillus subtilis trp leader region RNA causes efficient transcription termination at a weak intrinsic terminator

Complex regulation of the global regulatory gene csrA: CsrA‐mediated translational repression, transcription from five promoters by Eσ⁷⁰ and Eσ^S, and indirect transcriptional activation by CsrA

TRAP-5′ stem–loop interaction increases the efficiency of transcription termination in the Bacillus subtilis trpEDCFBA operon leader region

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Role of RNA Structure in Transcription Attenuation in Bacillus subtilis: The trpEDCFBA Operon as a Model System

Cited by 17 publications

References 14 publications

TRAP binding to the Bacillus subtilis trp leader region RNA causes efficient transcription termination at a weak intrinsic terminator

TRAP binding to the Bacillus subtilis trp leader region RNA causes efficient transcription termination at a weak intrinsic terminator

Complex regulation of the global regulatory gene csrA: CsrA‐mediated translational repression, transcription from five promoters by Eσ70 and EσS, and indirect transcriptional activation by CsrA

TRAP-5′ stem–loop interaction increases the efficiency of transcription termination in the Bacillus subtilis trpEDCFBA operon leader region

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Complex regulation of the global regulatory gene csrA: CsrA‐mediated translational repression, transcription from five promoters by Eσ⁷⁰ and Eσ^S, and indirect transcriptional activation by CsrA