Complexity in Regulation of Tryptophan Biosynthesis in <i>Bacillus subtilis</i>

Gollnick, Paul; Babitzke, Paul; Antson, A.A.; Yanofsky, Charles

doi:10.1146/annurev.genet.39.073003.093745

Cited by 149 publications

(159 citation statements)

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“…Since no complexes were detected with 3, 6 or 9 AT subunits/TRAP 11mer, it was suggested that AT binds to TRAP as a preformed 12mer 30 . However, the concentrations of AT 3 used in these studies, 12-48 µM (with equal fractions of AT 3 and AT 12 seen at 20 µM), are three orders of magnitude higher than have been measured for AT in vivo (~30 nM AT 3 ) 31 , suggesting that AT may function as a trimer in vivo In crystals of AT, four timers further assemble into a dodecamer ( Figure 6B) 26 , although there is no evidence that this is a biologically relevant assembly of AT.…”

Section: Discussionmentioning

confidence: 99%

“…These residues are the best candidates for being involved in direct interactions with TRAP and substitution of these residues is unlikely to disrupt the structure of AT 3 . Residues with buried side chains or those involved in monomer-monomer interactions in AT 3 were not substituted so as to avoid disrupting the protein's structure. We also avoided changing the cysteines or glycines in the two CXXCXGXG Zn-binding domains ( Figure 1A) because previous studies have shown that the bound Zn is required for AT structure and function 28 .…”

Section: Alanine-scanning Of the Surface Residues Of Atmentioning

confidence: 99%

“…The Zn-binding arms and the bottom region of AT 3 AT contains two cysteine-rich CXXCXGXG motifs with sequence 4 and structural 26 similarity to the Zn-binding domain of the type I family of Hsp40 molecular chaperones ( Figure 1A ); or in the β-sheet (β 1 -β 4 ) between the Zn-binding domain and the α-helical core of the protein (Thr10 or Ile35), had little or no effect on AT binding to TRAP (Table 1 and Figure 2B). These observations suggest that the side chains of residues located on the Zn-binding arms of AT do not directly interact with TRAP.…”

Section: Residues On Top Of At Trimer Are Important For Binding Trapmentioning

confidence: 99%

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Alanine Scanning Mutagenesis of Anti-TRAP (AT) Reveals Residues Involved in Binding to TRAP

Chen

Gollnick

2008

Journal of Molecular Biology

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SUMMARYThe trp RNA-binding attenuation protein (TRAP) regulates expression of the tryptophan biosynthetic (trp) genes in response to changes in intracellular levels of free L-tryptophan in many gram positive bacteria. When activated by binding tryptophan, TRAP binds to the mRNAs of several genes involved in tryptophan metabolism, and down-regulates transcription or translation of these genes. Anti-TRAP (AT) is an antagonist of TRAP that binds to tryptophan-activated TRAP and prevents it from binding to its RNA targets, and thereby up-regulates trp gene expression. The crystal structure shows that AT is a cone-shaped trimer (AT 3 ) with the N-terminal residues of the three subunits assembled at the apex of the cone and that these trimers can further assemble into a dodecameric (AT 12 ) structure. Using alanine-scanning mutagenesis we found four residues, all located on the "top" region of AT 3 , which are essential for binding to TRAP. Fluorescent labeling experiments further suggest that the top region of AT is in close juxtaposition to TRAP in the AT-TRAP complex. In vivo studies confirmed the importance of these residues on the top of AT in regulating TRAP mediated gene regulation.

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Section: Discussionmentioning

confidence: 99%

Section: Alanine-scanning Of the Surface Residues Of Atmentioning

confidence: 99%

Section: Residues On Top Of At Trimer Are Important For Binding Trapmentioning

confidence: 99%

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Alanine Scanning Mutagenesis of Anti-TRAP (AT) Reveals Residues Involved in Binding to TRAP

Chen

Gollnick

2008

Journal of Molecular Biology

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“…While trpG is present in an operon primarily concerned with folic acid biosynthesis (Fig. 1A), the TrpG polypeptide functions as the common glutamine amidotransferase subunit of anthranilate synthase (TrpE-TrpG) and para-aminobenzoate synthase (PabB-TrpG) in tryptophan and folic acid biosynthesis, respectively (13). The finding that trpG is regulated less tightly than trpP, despite having a higher affinity for TRAP (Tables 2 and 3), combined with the arrangement of the pabB and trpG coding sequences with respect to the TRAP binding site (Fig.…”

Section: Discussionmentioning

confidence: 99%

Translation Control of trpG from Transcripts Originating from the Folate Operon Promoter of Bacillus subtilis Is Influenced by Translation-Mediated Displacement of Bound TRAP, While Translation Control of Transcripts Originating from a Newly Identified trpG Promoter Is Not

Yakhnin

Babitzke

2007

J Bacteriol

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Bacillus subtilis trpG encodes a glutamine amidotransferase subunit that participates in the biosynthesis of both tryptophan and folic acid. TRAP inhibits translation of trpG in response to tryptophan by binding to a site that overlaps the trpG Shine-Dalgarno sequence, thereby blocking ribosome binding. Similar mechanisms regulate trpP and ycbK translation. The equilibrium binding constants of tryptophan-activated TRAP for the trpG, ycbK, and trpP transcripts were determined to be 8, 3, and 50 nM, respectively. Despite TRAP having a higher affinity for the trpG transcript, TRAP exhibited the least control of trpG expression. The trpG ShineDalgarno sequence overlaps the stop codon of the upstream pabB gene, while six of nine triplet repeats within the TRAP binding site are located upstream of the pabB stop codon. Thus, ribosomes translating the upstream pabB cistron could be capable of reducing TRAP-dependent control of TrpG synthesis by displacing bound TRAP. Expression studies using pabB-trpG -lacZ fusions in the presence or absence of an engineered stop codon within pabB suggest that translation-mediated displacement of bound TRAP reduces TRAP-dependent inhibition of TrpG synthesis from transcripts originating from the folate operon promoter (P pabB ). A new trpG promoter (P trpG ) was identified in the pabB coding sequence that makes a larger contribution to trpG expression than does P pabB . We found that TRAP-dependent regulation of trpG expression is more extensive for a transcript originating from P trpG and that transcripts originating from P trpG are not subject to translationmediated displacement of bound TRAP.

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“…In contrast, during tryptophan-replete conditions the translating ribosome masks sequence elements required for antiterminator formation, instead allowing for the mutually exclusive formation of an intrinsic terminator. In other microorganisms, a tryptophan-binding protein (TRAP) associates with a different regulatory RNA in order to control transcription attenuation or translation initiation of downstream tryptophan biosynthesis genes [10]. In addition to these mechanisms, a class of regulatory RNAs termed T-box RNAs specifically associates with cognate uncharged tRNAs in order to promote expression of downstream amino acid biosynthesis and transport genes [11].…”

Section: Riboswitches: Remnants Of An Ancient Mode Of Genetic Regulatmentioning

confidence: 99%

Structural features of metabolite-sensing riboswitches

Wakeman

Winkler

Dann

2007

Trends in Biochemical Sciences

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Riboswitches, metabolite-sensing RNA elements found in untranslated regions of the transcripts they regulate, possess extensive tertiary structure to couple metabolite binding to genetic control. Herein, we discuss recently published structures from four riboswitch classes and compare these natural RNA structures to those of in vitro selected RNA aptamers, which bind similar ligands to those of the riboswitches. Additionally, we examine the glmS riboswitch, the first example of a ribozyme-based riboswitch. This RNA provides the latest twist in the riboswitch field and portends exciting advances in the coming years. Our knowledge of the mechanisms of genetic regulation by riboswitches has increased mightily in recent years and will continue to grow as new riboswitch classes and ligands are discovered and structurally characterized. KeywordsRNA structure; riboswitch; ribozyme; transcription attenuation; noncoding RNAs; posttranscriptional regulation; crystallography; NMR Riboswitches: Remnants of an ancient mode of genetic regulation?The "central dogma" states that information is stored as DNA, transcribed into RNA, and translated into proteins, which are traditionally thought to satisfy most cellular structural and catalytic requirements. Genetic control of these steps is believed to occur primarily through the action of regulatory proteins; however, the importance of noncoding RNAs in these processes has become increasingly appreciated in recent years [1]. In eubacterial organisms, regulatory RNAs can elicit control of gene expression through regulation of transcription attenuation, translation initiation [reviewed in 2,3], or mRNA stability [4]. These eubacterial regulatory RNAs function via diverse intermolecular (trans) or intramolecular (cis) mechanisms to regulate the target mRNA. Trans-acting regulatory RNAs interact with target mRNAs to control translation, mRNA stability, or transcription termination [reviewed in 5,6, 7], whereas others regulate gene expression through specific sequestration of RNA-binding proteins [8]. Cis-acting RNAs, which are the focus of this review, are located within untranslated regions (UTRs) of the mRNA transcript that they regulate. Genetic control by the latter RNA elements can be accomplished either through the sole action of the RNA molecule or in conjunction with recruited protein factors.A classic example of a cis-acting regulatory RNA is the transcription attenuation control of tryptophan biosynthesis genes in certain Gram-negative bacteria [9]. For this mechanism, the kinetics of translation of a short leader peptide dictates the conformational state of the overall 5′-UTR. Under tryptophan-depleted conditions the translating ribosome stalls at tryptophan codons within the leader peptide thereby allowing for formation of an antiterminator helix and [24-26; reviewed in 27,12]. Given their ability to function as sensitive sentinels for intracellular metabolites in a protein-independent manner, these RNAs are likely to require exquisite structural sophistication....

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Complexity in Regulation of Tryptophan Biosynthesis in Bacillus subtilis

Cited by 149 publications

References 65 publications

Alanine Scanning Mutagenesis of Anti-TRAP (AT) Reveals Residues Involved in Binding to TRAP

Alanine Scanning Mutagenesis of Anti-TRAP (AT) Reveals Residues Involved in Binding to TRAP

Translation Control of trpG from Transcripts Originating from the Folate Operon Promoter of Bacillus subtilis Is Influenced by Translation-Mediated Displacement of Bound TRAP, While Translation Control of Transcripts Originating from a Newly Identified trpG Promoter Is Not

Structural features of metabolite-sensing riboswitches

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