The Interaction of RNA with TRAP: The Role of Triplet Repeats and Separating Spacer Nucleotides

Hopcroft, N.H.; Manfredo, Amanda; Wendt, Alice; Brzozowski, A.M.; Gollnick, Paul; Antson, A.A.

doi:10.1016/j.jmb.2004.02.038

Cited by 35 publications

(51 citation statements)

References 26 publications

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“…Binding of tryptophan leads to a reordering of 11 cationic surface domains (KKR motifs), allowing these regions to interact with trp leader RNA (Antson et al 1995(Antson et al , 1999Yang et al 1997;McElroy et al 2006). The TRAP-binding site in the trp leader transcript contains 11 (G/U)AG triplet repeats separated by two or three nonconserved spacer nucleotides (Babitzke et al , 1996Hopcroft et al 2004). Since six of the triplet repeats lie within an antiterminator structure that promotes transcriptional readthrough, TRAP binding prevents formation of this structure, which allows formation of a mutually exclusive terminator hairpin (Fig.…”

Section: Introductionmentioning

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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Section: Introductionmentioning

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 is a toroid-shaped molecule composed of 11 identical subunits with 11 tryptophan-binding sites located at subunitsubunit interfaces (5,6). In TRAP͞RNA complexes, the 11 (G͞U)AG repeats of the RNA chain bind to 11 binding sites on the surface of the protein and form a belt around the protein ring (23)(24)(25). A study with mutant TRAP proteins revealed that AT forms a complex only with tryptophan-activated TRAP and appears to recognize a region that is also involved in RNA binding (19).…”

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confidence: 99%

Crystal structure of Bacillus subtilis anti-TRAP protein, an antagonist of TRAP/RNA interaction

Shevtsov

Chen

Gollnick

et al. 2005

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

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In Bacillus subtilis the anti-TRAP protein (AT) is produced in response to the accumulation of uncharged tRNA Trp . AT regulates expression of genes involved in tryptophan biosynthesis and transport by binding to the tryptophan-activated trp RNA-binding attenuation protein (TRAP) and preventing its interaction with several mRNAs. Here, we report the x-ray structure of AT at 2.8 Å resolution, showing that the protein subunits assemble into tight trimers. Four such trimers are further associated into a 12-subunit particle in which individual trimers are related by twofold and threefold symmetry axes. Twelve DnaJ-like, cysteine-rich zincbinding domains form spikes on the surface of the dodecamer. Available data suggest several possible ways for AT to interact with the 11-subunit TRAP. Interaction between the two symmetrymismatching molecules could be assisted by the flexible nature of AT zinc-binding domains.DnaJ ͉ trp RNA-binding attenuation protein ͉ tryptophan biosynthesis ͉ protein-protein interactions

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“…Although GAG repeats are preferred, UAG repeats, such as in the first triplet repeat, occur when additional flexibility of the RNA is beneficial (Hopcroft et al 2004). This correlation is also observed with the fifth (UAG) and sixth (UAG) triplet repeats, which flank a suboptimal GG spacer.…”

Section: Discussionmentioning

confidence: 99%

“…The crystal structure of Bacillus stearothermophilus TRAP complexed with a synthetic RNA target indicates that Lys37, Lys56, and Arg58 (KKR) form hydrogen bonds with the A and the G residues in the second and third positions of the triplet repeat (Antson et al 1999;Elliott et al 1999). Gly18 and Asp39 form additional hydrogen bonds with the triplet repeats, and Phe32 likely participates in a stacking interaction (Hopcroft et al 2004). Few contacts form between TRAP and the first nucleotide of the triplet, which explains the sequence variability at this position.…”

Section: Introductionmentioning

confidence: 98%

“…Few contacts form between TRAP and the first nucleotide of the triplet, which explains the sequence variability at this position. No direct interactions occur between TRAP and the nucleotides separating the triplet repeats, although the sequence of these spacers can influence TRAP binding (Babitzke et al , 1996Hopcroft et al 2004). Since the KKR motifs encircle TRAP, the single-stranded triplet repeat region wraps around the perimeter of the protein upon binding (Fig.…”

Section: Introductionmentioning

confidence: 99%

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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 Interaction of RNA with TRAP: The Role of Triplet Repeats and Separating Spacer Nucleotides

Cited by 35 publications

References 26 publications

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

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

Crystal structure of Bacillus subtilis anti-TRAP protein, an antagonist of TRAP/RNA interaction

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

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