The mtrB gene of Bacillus pumilus encodes a protein with sequence and functional homology to the trp RNA-binding attenuation protein (TRAP) of Bacillus subtilis

Hoffman, Robert J.; Gollnick, Paul

doi:10.1128/jb.177.3.839-842.1995

Cited by 23 publications

(26 citation statements)

References 19 publications

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“…3 and 4). We observed prominent CMCT signals at the U and G residues corresponding to positions 5,7,18,20,21,35,36,38, and 41, indicating that these residues are single stranded. The absence of CMCT modification of the remaining U and G residues suggests that these nucleotides are base paired ( Fig.…”

Section: Resultsmentioning

confidence: 88%

“…This finding is consistent with a previous in vivo study where it was determined that changing this sequence to GUG had little effect on trp operon expression (28). Note that, with the exception of the first UAG repeat, bound TRAP prevented or reduced cleavage of the G residues in the (G/U)AG repeats that were previously shown to interact with TRAP (data not shown) (18).…”

Section: Resultsmentioning

confidence: 99%

“…8B). The trp operon leader transcripts of Bacillus pumilus (18), Bacillus caldotenax (31), and B. stearothermophilus (29) also contain 5Ј stem-loops and multiple triplet repeats, as well as overlapping antiterminator and terminator structures. Thus, it appears that all four organisms control expression of the trp operon by essentially identical transcription attenuation mechanisms.…”

Section: Discussionmentioning

confidence: 99%

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trp RNA-Binding Attenuation Protein-5′ Stem-Loop RNA Interaction Is Required for Proper Transcription Attenuation Control of the Bacillus subtilis trpEDCFBA Operon

Yakhnin

Dharmaraj

et al. 2000

J Bacteriol

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The trp RNA-binding attenuation protein (TRAP) regulates expression of the Bacillus subtilis trpEDCFBA operon by a novel transcription attenuation mechanism. Tryptophan-activated TRAP binds to the nascent trp leader transcript by interacting with 11 (G/U)AG repeats, 6 of which are present in an antiterminator structure. TRAP binding to these repeats prevents formation of the antiterminator, thereby promoting formation of an overlapping intrinsic terminator. A third stem-loop structure that forms at the extreme 5 end of the trp leader transcript also plays a role in the transcription attenuation mechanism. The 5 stem-loop increases the affinity of TRAP for trp leader RNA. Results from RNA structure mapping experiments demonstrate that the 5 stem-loop consists of a 3-bp lower stem, a 5-by-2 asymmetric internal loop, a 6-bp upper stem, and a hexaloop at the apex of the structure. Footprinting results indicate that TRAP interacts with the 5 stem-loop and that this interaction differs depending on the number of downstream (G/U)AG repeats present in the transcript. Expression studies with trpE -lacZ translational fusions demonstrate that TRAP-5 stemloop interaction is required for proper regulation of the trp operon. 3 RNA boundary experiments indicate that the 5 structure reduces the number of (G/U)AG repeats required for stable TRAP-trp leader RNA association. Thus, TRAP-5 stem-loop interaction may increase the likelihood that TRAP will bind to the (G/U)AG repeats in time to block antiterminator formation.Expression of the Bacillus subtilis tryptophan biosynthetic genes is regulated in response to changes in the intracellular level of tryptophan by the trp RNA-binding attenuation protein (TRAP) (4, 16). The trpEDCFBA operon is regulated by TRAP-mediated transcription attenuation (5,10,17,19,24,25) and translational control mechanisms (14,19,22). TRAP also regulates expression of the unlinked trpG gene at the translational level (8,15,30). TRAP exists as a complex consisting of 11 identical subunits arranged in a single ring termed the ␤-wheel (1, 3). Tryptophan cooperatively activates TRAP by binding between every adjacent TRAP subunit (3, 6).The 203-nucleotide untranslated trp operon leader transcript can fold into three distinct RNA secondary structures that participate in transcription attenuation (Fig. 1). When TRAP is activated by tryptophan, 11 KKR motifs that outline the periphery of the TRAP complex can bind to 11 closely spaced (G/U)AG repeats present in the nascent trp leader transcript, thereby wrapping the RNA around the periphery of the TRAP complex (2, 8, 31). TRAP binding blocks formation of the antiterminator since six of the (G/U)AG repeats are present within this RNA structure (5, 8). Thus, TRAP binding promotes formation of the overlapping intrinsic terminator which results in transcription termination before RNA polymerase can reach the trp operon structural genes. In the absence of TRAP binding, formation of the antiterminator permits transcription of the entire operon (5).While it is not known ...

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Section: Resultsmentioning

confidence: 88%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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trp RNA-Binding Attenuation Protein-5′ Stem-Loop RNA Interaction Is Required for Proper Transcription Attenuation Control of the Bacillus subtilis trpEDCFBA Operon

Yakhnin

Dharmaraj

et al. 2000

J Bacteriol

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“…The pnpA mutant was BG119, a derivative of BG1 in which an internal portion of the pnpA gene has been replaced with a kanamycin resistance gene (11). Some of the control experiments for TRAP-specific binding in the protein extract were done with BG4233, an mtrB deletion mutant (12).…”

Section: Methodsmentioning

confidence: 99%

Recycling of a regulatory protein by degradation of the RNA to which it binds

Deikus

Babitzke

Bechhofer

2004

Proc. Natl. Acad. Sci. U.S.A.

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When Bacillus subtilis is grown in the presence of excess tryptophan, transcription of the trp operon is regulated by binding of tryptophan-activated TRAP to trp leader RNA, which promotes transcription termination in the trp leader region. Transcriptome analysis of a B. subtilis strain lacking polynucleotide phosphorylase (PNPase; a 3 -to-5 exoribonuclease) revealed a striking overexpression of trp operon structural genes when the strain was grown in the presence of abundant tryptophan. Analysis of trp leader RNA in the PNPase ؊ strain showed accumulation of a stable, TRAPprotected fragment of trp leader RNA. Loss of trp operon transcriptional regulation in the PNPase ؊ strain was due to the inability of ribonucleases other than PNPase to degrade TRAP-bound leader RNA, resulting in the sequestration of limiting TRAP. Thus, in the case of the B. subtilis trp operon, specific ribonuclease degradation of RNA in an RNA-protein complex is required for recycling of an RNA-binding protein. Such a mechanism may be relevant to other systems in which limiting concentrations of an RNA-binding protein must keep pace with ongoing transcription.T ranscription attenuation is a form of gene regulation in which transcription of a gene or operon is regulated by the folding of a leader RNA to form either a transcription terminator, thus preventing transcription of downstream structural genes, or an antiterminator, thus allowing transcription to proceed (1, 2). Regulated expression of structural genes in the trp operon of several bacteria has been fertile ground for elucidating various transcription attenuation mechanisms. In Escherichia coli, the trp leader terminator structure is formed when tryptophan is abundant, and the antiterminator structure forms when tryptophan is scarce, because of ribosome stalling at tryptophan codons in the leader peptide coding sequence. In Lactococcus lactis, it is likely that formation of the antiterminator structure depends on binding of uncharged tRNA Trp to leader RNA (the ''T-box'' mechanism) (1, 2). In both of these cases, the terminator structure is the default; the antiterminator structure, which allows transcription of trp structural genes, is formed only in response to a scarcity of tryptophan. By contrast, in Bacillus subtilis the trp leader antiterminator structure is the default. Formation of the terminator structure depends on binding of 1 TRAP, a regulatory protein that can bind trp leader RNA only when it is activated by tryptophan (3-5). In conditions of low tryptophan, TRAP does not bind trp leader RNA, allowing formation of the antiterminator structure and transcription of the trp operon structural genes. When tryptophan is abundant, a TRAP 11-mer is activated, which can then bind to 11 trinucleotide repeats in the trp leader RNA, resulting in the formation of the terminator structure (6, 7).There is a long-standing question regarding TRAP-mediated regulation of the B. subtilis trp operon: How can a limited amount of TRAP suffice to keep pace with ongoing transcription from the t...

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“…These nucleotides are strongly conserved in 59SL structures among organisms closely related to B. subtilis, raising the possibility that the 59SL participates in the trp operon attenuation mechanisms in these organisms as well. However, with the exception of Bacillus pumilus (Hoffman and Gollnick 1995) and Bacillus halodurans (Szigeti et al 2004), the presumed transcription attenuation mechanisms of these other organisms have not been experimentally characterized.…”

Section: Introductionmentioning

confidence: 99%

Molecular basis of TRAP–5′SL RNA interaction in the Bacillus subtilis trp operon transcription attenuation mechanism

McGraw¹,

Mokdad²,

Major³

et al. 2008

RNA

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Expression of the Bacillus subtilis trpEDCFBA operon is regulated by the interaction of tryptophan-activated TRAP with 11 (G/U)AG trinucleotide repeats that lie in the leader region of the nascent trp transcript. Bound TRAP prevents folding of an antiterminator structure and favors formation of an overlapping intrinsic terminator hairpin upstream of the trp operon structural genes. A 59-stem-loop (59SL) structure that forms just upstream of the triplet repeat region increases the affinity of TRAP-trp RNA interaction, thereby increasing the efficiency of transcription termination. Single-stranded nucleotides in the internal loop and in the hairpin loop of the 59SL are important for TRAP binding. We show here that altering the distance between these two loops suggests that G7, A8, and A9 from the internal loop and A19 and G20 from the hairpin loop constitute two structurally discrete TRAP-binding regions. Photochemical cross-linking experiments also show that the hairpin loop of the 59SL is in close proximity to the flexible loop region of TRAP during TRAP-59SL interaction. The dimensions of B. subtilis TRAP and of a three-dimensional model of the 59SL generated using the MC-Sym and MC-Fold pipeline imply that the 59SL binds the protein in an orientation where the helical axis of the 59SL is perpendicular to the plane of TRAP. This interaction not only increases the affinity of TRAP-trp leader RNA interaction, but also orients the downstream triplet repeats for interaction with the 11 KKR motifs that lie on TRAP's perimeter, increasing the likelihood that TRAP will bind in time to promote termination.

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The mtrB gene of Bacillus pumilus encodes a protein with sequence and functional homology to the trp RNA-binding attenuation protein (TRAP) of Bacillus subtilis

Cited by 23 publications

References 19 publications

trp RNA-Binding Attenuation Protein-5′ Stem-Loop RNA Interaction Is Required for Proper Transcription Attenuation Control of the Bacillus subtilis trpEDCFBA Operon

trp RNA-Binding Attenuation Protein-5′ Stem-Loop RNA Interaction Is Required for Proper Transcription Attenuation Control of the Bacillus subtilis trpEDCFBA Operon

Recycling of a regulatory protein by degradation of the RNA to which it binds

Molecular basis of TRAP–5′SL RNA interaction in the Bacillus subtilis trp operon transcription attenuation mechanism

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