Uncharged tRNA Trp Research Articles

Solution structure, dynamics and tetrahedral assembly of Anti-TRAP, a homo-trimeric triskelion-shaped regulator of tryptophan biosynthesis in Bacillus subtilis

Cellular production of tryptophan is metabolically expensive and tightly regulated. The small Bacillus subtilis zinc binding Anti-TRAP protein (AT), which is the product of the yczA/rtpA gene, is upregulated in response to accumulating levels of uncharged tRNATrp through a T-box antitermination mechanism. AT binds to the undecameric axially symmetric ring-shaped protein TRAP (trp RNA Binding Attenuation Protein), thereby preventing it from binding to the trp leader RNA. This reverses the inhibitory effect of TRAP on transcription and translation of the trp operon. AT principally adopts two symmetric oligomeric states, a trimer (AT3) featuring three-fold axial symmetry or a dodecamer (AT12) comprising a tetrahedral assembly of trimers, whereas only the trimeric form binds and inhibits TRAP. We apply native mass spectrometry (nMS) and small-angle x-ray scattering (SAXS), together with analytical ultracentrifugation (AUC) to monitor the pH and concentration-dependent equilibrium between the trimeric and dodecameric structural forms of AT. In addition, we use solution nuclear magnetic resonance (NMR) spectroscopy to determine the solution structure of AT3, while heteronuclear 15N relaxation measurements on both oligomeric forms of AT provide insights into the dynamic properties of binding-active AT3 and binding-inactive AT12, with implications for TRAP binding and inhibition.

Positions of Trp Codons in the Leader Peptide-Coding Region of the at Operon Influence Anti-Trap Synthesis and trp Operon Expression in Bacillus licheniformis

Tryptophan, phenylalanine, tyrosine, and several other metabolites are all synthesized from a common precursor, chorismic acid. Since tryptophan is a product of an energetically expensive biosynthetic pathway, bacteria have developed sensing mechanisms to downregulate synthesis of the enzymes of tryptophan formation when synthesis of the amino acid is not needed. In Bacillus subtilis and some other Gram-positive bacteria, trp operon expression is regulated by two proteins, TRAP (the tryptophan-activated RNA binding protein) and AT (the anti-TRAP protein). TRAP is activated by bound tryptophan, and AT synthesis is increased upon accumulation of uncharged tRNA(Trp). Tryptophan-activated TRAP binds to trp operon leader RNA, generating a terminator structure that promotes transcription termination. AT binds to tryptophan-activated TRAP, inhibiting its RNA binding ability. In B. subtilis, AT synthesis is upregulated both transcriptionally and translationally in response to the accumulation of uncharged tRNA(Trp). In this paper, we focus on explaining the differences in organization and regulatory functions of the at operon's leader peptide-coding region, rtpLP, of B. subtilis and Bacillus licheniformis. Our objective was to correlate the greater growth sensitivity of B. licheniformis to tryptophan starvation with the spacing of the three Trp codons in its at operon leader peptide-coding region. Our findings suggest that the Trp codon location in rtpLP of B. licheniformis is designed to allow a mild charged-tRNA(Trp) deficiency to expose the Shine-Dalgarno sequence and start codon for the AT protein, leading to increased AT synthesis.

Physiological Effects of Anti-TRAP Protein Activity and tRNA Trp Charging on trp Operon Expression in Bacillus subtilis

The Bacillus subtilis anti-TRAP protein regulates the ability of the tryptophan-activated TRAP protein to bind to trp operon leader RNA and promote transcription termination. AT synthesis is regulated both transcriptionally and translationally by uncharged tRNA(Trp). In this study, we examined the roles of AT synthesis and tRNA(Trp) charging in mediating physiological responses to tryptophan starvation. Adding excess phenylalanine to wild-type cultures reduced the charged tRNA(Trp) level from 80% to 40%; the charged level decreased further, to 25%, in an AT-deficient mutant. Adding tryptophan with phenylalanine increased the charged tRNA(Trp) level, implying that phenylalanine, when added alone, reduces the availability of tryptophan for tRNA(Trp) charging. Changes in the charged tRNA(Trp) level observed during growth with added phenylalanine were associated with increased transcription of the genes of tryptophan metabolism. Nutritional shift experiments, from a medium containing tryptophan to a medium with phenylalanine and tyrosine, showed that wild-type cultures gradually reduced their charged tRNA(Trp) level. When this shift was performed with an AT-deficient mutant, the charged tRNA(Trp) level decreased even further. Growth rates for wild-type and mutant strains deficient in AT or TRAP or that overproduce AT were compared in various media. A lack of TRAP or overproduction of AT resulted in phenylalanine being required for growth. These findings reveal the importance of AT in maintaining a balance between the synthesis of tryptophan versus the synthesis of phenylalanine, with the level of charged tRNA(Trp) acting as the crucial signal regulating AT production.

Two conformations of a crystalline human tRNA synthetase–tRNA complex: implications for protein synthesis

Aminoacylation of tRNA is the first step of protein synthesis. Here, we report the co-crystal structure of human tryptophanyl-tRNA synthetase and tRNATrp. This enzyme is reported to interact directly with elongation factor 1alpha, which carries charged tRNA to the ribosome. Crystals were generated from a 50/50% mixture of charged and uncharged tRNATrp. These crystals captured two conformations of the complex, which are nearly identical with respect to the protein and a bound tryptophan. They are distinguished by the way tRNA is bound. In one, uncharged tRNA is bound across the dimer, with anticodon and acceptor stem interacting with separate subunits. In this cross-dimer tRNA complex, the class I enzyme has a class II-like tRNA binding mode. This structure accounts for biochemical investigations of human TrpRS, including species-specific charging. In the other conformation, presumptive aminoacylated tRNA is bound only by the anticodon, the acceptor stem being free and having space to interact precisely with EF-1alpha, suggesting that the product of aminoacylation can be directly handed off to EF-1alpha for the next step of protein synthesis.

Complexity in Regulation of Tryptophan Biosynthesis in Bacillus subtilis

Bacillus subtilis uses novel regulatory mechanisms in controlling expression of its genes of tryptophan synthesis and transport. These mechanisms respond to changes in the intracellular concentrations of free tryptophan and uncharged tRNA(Trp). The major B. subtilis protein that regulates tryptophan biosynthesis is the tryptophan-activated RNA-binding attenuation protein, TRAP. TRAP is a ring-shaped molecule composed of 11 identical subunits. Active TRAP binds to unique RNA segments containing multiple trinucleotide (NAG) repeats. Binding regulates both transcription termination and translation in the trp operon, and translation of other coding regions relevant to tryptophan metabolism. When there is a deficiency of charged tRNA(Trp), B. subtilis forms an anti-TRAP protein, AT. AT antagonizes TRAP function, thereby increasing expression of all the genes regulated by TRAP. Thus B. subtilis and Escherichia coli respond to identical regulatory signals, tryptophan and uncharged tRNA(Trp), yet they employ different mechanisms in regulating trp gene expression.

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

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 angstroms 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 zinc-binding 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 symmetry-mismatching molecules could be assisted by the flexible nature of AT zinc-binding domains.

Interaction of the trp RNA-binding Attenuation Protein (TRAP) with Anti-TRAP

The trp RNA-binding attenuation protein (TRAP) negatively regulates expression of the tryptophan biosynthesis genes of Bacillus subtilis. In the presence of tryptophan, TRAP is activated to bind to the 5′-leader region of the trp mRNA resulting in termination prior to the structural genes. In addition, accumulation of uncharged tRNA Trp induces synthesis of anti-TRAP (AT), which binds to TRAP and inhibits its function. Both of these proteins consist of oligomers of identical subunits. Here, we characterize the self-association of each of these proteins and the TRAP–AT interaction in free solution using equilibrium and velocity analytical ultracentrifugation. TRAP exists as a stable 11-mer in the absence and in the presence of tryptophan. Tryptophan binding induces a conformational change in TRAP. AT exists in a reversible equilibrium between trimer and dodecamer with an equilibrium constant of ∼3×10 14 M −3. About 20% of the trimer is incompetent to form dodecamer. The AT equilibrium is slow on the time-scale of the velocity experiment. Formation of TRAP–AT complexes occurs only in the presence of tryptophan. A complex containing one TRAP 11-mer and one AT 12-mer forms with high affinity. At higher ratios of TRAP:AT complexes containing two TRAP 11-mers and one AT 12-mer are detected. A model for the structure of the complex is proposed.

A Double Dose of Trp Regulation

Amino acid synthesis is tightly controlled in bacteria by a system in which the amino acids themselves, as well as their corresponding transfer RNAs (tRNAs), regulate production. For example, in Bacillus subtilis , the mechanism for regulating tryptophan (Trp) synthesis involves an antiterminator protein called TRAP, which is activated by Trp. However, TRAP activity is antagonized by a protein called anti-TRAP (AT), where the synthesis of AT is induced by uncharged tRNA Trp . Chen and Yanofsky now show another level of regulation for the Trp operon in which uncharged tRNA Trp also regulates the translation of AT through tandem Trp codons in a leader peptide coding sequence. This mechanism is somewhat reminiscent of the classical Trp attenuation system in Escherchia coli ; however, the leader peptide in B. subtilis exerts its effect at the level of translation instead of transcription termination, as seen in E. coli . The authors term the B. subtilis system "regulatory sophistication" because it shows both transcriptional and translational regulation of AT in response to tRNA Trp . G. Chen, C. Yanofsky, Tandem transcription and translation regulatory sensing of uncharged tryptophan tRNA. Science 301 , 211-213 (2003). [Abstract] [Full Text]

Tandem transcription and translation regulatory sensing of uncharged tryptophan tRNA.

The Bacillus subtilis AT (anti-TRAP) protein inhibits the regulatory protein TRAP (trp RNA-binding attenuation protein), thereby eliminating transcription termination in the leader region of the trp operon. Transcription of the AT operon is activated by uncharged tryptophan transfer RNA (tRNATrp). Here we show that translation of AT also is regulated by uncharged tRNATrp. A 10-residue coding region containing three consecutive tryptophan codons is located immediately preceding the AT structural gene. Completion of translation of this coding region inhibits AT synthesis, whereas incomplete translation increases AT production. Tandem sensing of uncharged tRNATrp therefore regulates synthesis of AT, which in turn regulates TRAP's ability to inhibit trp operon expression.

Inhibition of the B. subtilis regulatory protein TRAP by the TRAP-inhibitory protein, AT.

An anti-TRAP (AT) protein, a factor of previously unknown function, conveys the metabolic signal that the cellular transfer RNA for tryptophan (tRNATrp) is predominantly uncharged. Expression of the operon encoding AT is induced by uncharged tRNATrp. AT associates with TRAP, the trp operon attenuation protein, and inhibits its binding to its target RNA sequences. This relieves TRAP-mediated transcription termination and translation inhibition, increasing the rate of tryptophan biosynthesis. AT binds to TRAP primarily when it is in the tryptophan-activated state. The 53-residue AT polypeptide is homologous to the zinc-binding domain of DnaJ. The mechanisms regulating tryptophan biosynthesis in Bacillus subtilis differ from those used by Escherichia coli.

TRNATrp as a key element of antitermination in the Lactococcus lactis trp operon.

The expression of the trp operon of Lactococcus lactis is regulated in response to tryptophan availability by a mechanism of transcription antitermination. We present evidence in support of a previously described model involving tRNATrp as a key element in the sensing of tryptophan levels and the realization of the regulatory response to tryptophan limitation. In agreement with this model, two sites of presumed direct interaction between the trp leader transcript and tRNATrp are found to be of crucial importance for efficient antitermination. These correspond to the specifier codon, which presumably interacts with the anticodon in the tRNA, and a sequence complementary to, and presumably interacting with, the acceptor stem of the tRNA. Through these interactions, uncharged tRNATrp is believed to stabilize an antiterminator conformation of the trp leader transcript, thus allowing transcription and expression of the structural genes of the operon. For the first time, we present direct evidence that it is the ratio of uncharged to charged tRNA that is important for the regulation of antitermination, rather than the absolute amount of uncharged tRNA. In addition, our results indicate that the codon-anticodon interaction, although contributing largely to the efficiency of the regulatory response, is not strictly indispensable, which suggests the existence of additional interactions between mRNA and tRNA. Finally, we describe a possible additional level of regulation, superimposed and dependent on tRNA-mediated anti-termination control, that is based on the processing of the trp leader transcript. Together with the regulation mechanisms described earlier for the Escherichia coli and Bacillus subtilis trp operons, this constitutes the third different mechanism of transcript elongation control found to be involved in the regulation of an operon of which the structural genes are highly conserved.

Some novel transcription attenuation mechanisms used by bacteria

A variety of transcription attenuation mechanisms are used by bacteria to regulate gene and operon expression. This review summarizes previous and current studies designed to elucidate the features of the specific attenuation mechanisms that regulate expression of the tryptophanase ( tna) operon of Escherichia coli and the tryptophan ( trp) operon of Bacillus subtilis. Initiation of transcription in the tna operon is regulated by catabolite repression. Once initiated, transcription is regulated by tryptophan-induced inhibition of Rho-mediated transcription termination in the leader region of the operon. An operon-encoded leader peptide, TnaC, containing a crucial tryptophan residue, plays an essential role in induction. This peptide appears to act in cis on the ribosome translating tnaC to inhibit its release at the tnaC stop codon. The stalled ribosome would block Rho's access to the tna transcript, thereby preventing termination. Transcription of the trp operon of B subtilis is regulated by an attenuation mechanism that responds to a tryptophan-activated eleven subunit RNA-binding regulatory protein, called TRAP. Activated TRAP binds to repeated GAG sequences in the leader segment of the trp operon transcript, disrupting an RNA antiterminator and promoting formation of a terminator. Activated TRAP also regulates translation of trpG in the folate operon by binding to repeat GAG sequences surrounding the trpG ribosome binding site. A temperature sensitive tryptophanyl-tRNA synthetase ( trpS) mutant was previously observed to overexpress the trp operon and trpG, when grown at elevated temperatures in the presence of tryptophan. We have found that the trpS defect increases trp operon and trpG expression by interfering with TRAP's ability to act. We suggest that either accumulation of uncharged tRNA Trp or overproduction of a TRAP-binding transcript reduces the level of functional TRAP in the trpS mutant.

Relationship between protein synthesis and concentrations of charged and uncharged tRNATrp in Escherichia coli.

We have continuously monitored Trp-tRNA(Trp) concentrations in vivo and, in the same cultures, measured rates of protein synthesis in isogenic stringent and relaxed strains. We have also manipulated cellular charged and uncharged [tRNA(Trp)] by two means: (i) the strain used contains a Trp-tRNA synthetase mutation that increases the Km for Trp; thus, varying exogenous Trp varies cellular Trp-tRNA(Trp); and (ii) we have introduced into the mutant strain a plasmid containing the tRNA(Trp) gene behind an inducible promoter; thus, total [tRNA(Trp)] also can be varied depending on length of induction. The use of these conditions, combined with a previously characterized assay system, has allowed us to demonstrate that (i) the rate of incorporation of Trp into protein is proportional to the fraction of tRNA(Trp) that is charged; for any given total [tRNA(Trp)], this rate is also proportional to the [Trp-tRNA(Trp)]; (ii) uncharged tRNA(Trp) inhibits incorporation of Trp into protein; and (iii) rates of incorporation into protein of at least two other amino acids, Lys and Cys, are also sensitive to [Trp-tRNA(Trp)] and are inhibited by uncharged tRNA(Trp). Our results are consistent with models of translational control that postulate modulating polypeptide chain elongation efficiency by varying concentrations of specific tRNAs.

Mutations affecting tRNA Trp and its charging and their effect on regulation of transcription termination at the attenuator of the tryptophan operon

A mutant altered in the sole structural gene for tRNA Trp produces sevenfold elevated levels of trp operon enzymes and messenger RNA. These increases are probably due to a reduced frequency of transcription termination at the attenuator region of the operon. A normal termination frequency is observed in this mutant, however, when conditions are imposed that should permit an increase in the extent of charging of the mutant tRNA Trp. Similarly, a mutant with an altered tryptophanyl-tRNA synthetase exhibits increased expression of the operon; this increase is also eliminated by conditions which should allow charging of a greater fraction of cellular tRNA Trp. These observations suggest that the extent of charging of tRNA Trp rather than the availability of tryptophan is the determinative factor in regulation at the attenuator. A second mutation which alters tRNA Trp, but which is not in the tRNA Trp structural gene, also increases expression of the operon. Conditions which should promote charging of this tRNA Trp do not overcome the regulatory defect. Analyses were performed with various trpT heterozygotes in an attempt to determine whether charged or uncharged tRNA Trp is the active species in the regulation of transcription termination at the attenuator. The findings obtained suggest that both species influence regulation at the attenuator.