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 approximately 3 x 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.     

Genetic fusions that help define a transcription termination region in Escherichia coli

The trpA gene product was analyzed from a class of strains of Escherichia coli K12 in which the lac operon has been fused by deletion to the trp operon. These are strains that have retained the ability to synthesize tryptophan. Two of these strains are shown to make a wild-type trpA product; these strains retain intact all structural genes of the ttrp operon. It is proposed that the lac operon in these strains is fused to a region of the trp operon between trpA, the last gene in the operon, and the region where trp messenger RNA synthesis terminates. The region where trp messenger RNA synthesis terminates thus is distinct from the trp structural genes.     

Dynamic allostery in the ring protein TRAP

We have discovered distinct, characteristic differences in the thermodynamic signatures of tryptophan binding by trp RNA-binding attenuation protein (TRAP) from two different bacterial species. The TRAP 11mer ring binds 11 molecules of tryptophan at symmetry-related sites. Tryptophan binding to Bacillus stearothermophilus TRAP is not cooperative, but isothermal titration calorimetry shows that filling the first tryptophan binding sites of Bacillus subtilis TRAP has a marked effect on the thermodynamics of subsequent ligand binding. We have identified a single, conservative amino acid replacement (Ile to Leu) in B. subtilis TRAP that abolishes this effect, and suggest the initial ligand binding causes a change throughout the wild-type protein ring.     

Thr region between the operator and first structural gene of the tryptophan operon of Escherichia coli may have a regulatory function

Among a collection of 34 independent mutants with internal deletions in the trp operon of Escherichia coli we found six that fail to recombine with any known point mutant in trpE, the first gene in the operon. These six deletion mutants are regulated normally by tryptophan and thus appear to have the trp operator region intact. However, four of these deletions result in alterations in the maximum level of expression of the trpC, B and A genes when compared with wild type or with an internal deletion of similar length which retains a small operatorproximal segment of trpE. Two of these deletion mutants, trpΔED1 and trpΔED12, have lower levels of the protein products of trpB and trpA than the control strains. In contrast, deletions trpΔED2 and trpΔED102 both markedly increase the levels of the trpB and trpA polypeptides. Deletion mutant trpΔED2 has 3 to 3.5 times and mutant trpΔED102 has seven to eight times as much tryptophan synthetase β₂ and α proteins as the wild-type or deletion control strains. The increase in tryptophan synthetase β₂ and α proteins seen is a consequence of an increase in the level of trp mRNA directing the synthesis of these enzymes. The rate of synthesis of trpBA mRNA is increased in trpΔAED2 about twofold, and in trpΔED102 about four- to sixfold over the control strain. The left-hand deletion end-points of both trpΔED2 and trpΔAED102 have been shown to map to the right of a known trp operator-constitutive mutation and appear to lie before the first translation start codon in trpE (M. Bronson, C. Squires &; C. Yanofsky, unpublished results). We propose that these deletions alter a region between the earliest known trpE point mutation and the trp operator which influences the maximum rate of synthesis of trp operon mRNA.     

Internal promoter of the tryptophan operon of Escherichia coli is located in a structural gene

The constitutive low-efficiency promoter site (P₂) near the middle of the tryptophan operon of Escherichia coli has been mapped by analysis of short deletions internal to the trp operon. Comparison of deletions which remove this internal promoter with those which retain it show that P₂ is located within trpD, the region coding for phosphoribosyl anthranilate transferase. P₂ maps near the operator-distal end of trpD, on the operator-proximal side of two trpD point mutants. Comparisons of strains with and without the P₂ site indicate that initiations at this promoter are responsible for synthesis of 80% of the trpC, trpB and trp A polypeptides present in repressed cells.     

Structural change in β-sheet A of Z α(1)-antitrypsin is responsible for accelerated polymerization and disease

The presence of the Z mutation (Glu342Lys) is responsible for more than 95% of α₁-antitrypsin (α₁AT) deficiency cases. It leads to increased polymerization of the serpin α₁AT during its synthesis and in circulation. It has been proposed that the Z mutation results in a conformational change within the folded state of antitrypsin that enhances its polymerization. In order to localize the conformational change, we have created two single tryptophan mutants of Z α₁AT and analyzed their fluorescence properties. α₁AT contains two tryptophan residues that are located in distinct regions of the molecule: Trp194 at the top of β-sheet A and Trp238 on β-sheet B. We have replaced each tryptophan residue individually with a phenylalanine in order to study the local environment of the remaining tryptophan residue in both M and Z α₁AT. A detailed fluorescence spectroscopic analysis of each mutant was carried out, and we detected differences in the emission spectrum, the Stern-Volmer constant for potassium iodide quenching and the anisotropy of only Trp194 in Z α₁AT compared to M α₁AT. Our data reveal that the Z mutation results in a conformational change at the top of β-sheet A but does not affect the structural integrity of β-sheet B.     

Polarity suppressors increase expression of the wild-type tryptophan operon of Escherichia coli

Three new polarity suppressors, selected to relieve the polar effect of nonsense mutations in the tryptophan (trp) and lactose (lac) operons of Escherichia coli, increase expression distal to nonsense mutations in both operons to a greater extent than suA. These suppressors relieve the polarity created by amber, ochre and frameshift mutations with equal efficiency.Two of the three polarity suppressors elevate enzyme synthesis in the wildtype trp operon two- and fivefold, respectively. The increase in enzyme levels is in each case correlated with increased levels and rates of synthesis of structural gene trp messenger RNA. Since expression of all genes is elevated, these findings suggest the existence of a site early in the wild-type trp operon that affects the extent of operon expression. We located the site affected by these two polarity suppressors between the operator and the first structural gene, trpE. Although the third polarity suppressor also relieves mutational polarity efficiently, it has no detectable effect on expression of the wild-type trp operon.     

Characterization of a trp RNA-binding attenuation protein (TRAP) mutant with tryptophan independent RNA binding activity

TRAP (trp RNA-binding attenuation protein) is an 11 subunit RNA-binding protein that regulates expression of genes involved in tryptophan metabolism (trp) in Bacillus subtilis in response to changes in intracellular tryptophan concentration. When activated by binding up to 11 tryptophan residues, TRAP binds to the mRNAs of several trp genes and down-regulates their expression. Recently, a TRAP mutant was found that binds RNA in the absence of tryptophan. In this mutant protein, Thr30, which is part of the tryptophan-binding site, is replaced with Val (T30V). We have compared the RNA-binding properties of T30V and wild-type (WT) TRAP, as well as of a series of hetero-11-mers containing mixtures of WT and T30V TRAP subunits. The most significant difference between the interaction of T30V and WT TRAP with RNA is that the affinity of T30V TRAP is more dependent on ionic strength. Analysis of the hetero-11-mers allowed us to examine how subunits interact within an 11-mer with regard to binding to tryptophan or RNA. Our data suggest that individual subunits retain properties similar to those observed when they are in homo-11-mers and that individual G/UAG triplets within the RNA can bind to TRAP differently.     

Substitutions of Thr30 provide mechanistic insight into tryptophan-mediated activation of TRAP binding to RNA

TRAP is an 11 subunit RNA binding protein that regulates expression of genes involved in tryptophan biosynthesis and transport in Bacillus subtilis. TRAP is activated to bind RNA by binding up to 11 molecules of l-tryptophan in pockets formed by adjacent subunits. The precise mechanism by which tryptophan binding activates TRAP is not known. Thr30 is in the tryptophan binding pocket. A TRAP mutant in which Thr30 is substituted with Val (T30V) does not bind tryptophan but binds RNA constitutively, suggesting that Thr30 plays a key role in the activation mechanism. We have examined the effects of other substitutions of Thr30. TRAP proteins with small beta-branched aliphatic side chains at residue 30 bind RNA constitutively, whereas those with a small polar side chain show tryptophan-dependent RNA binding. Several mutant proteins exhibited constitutive RNA binding that was enhanced by tryptophan. Although the tryptophan and RNA binding sites on TRAP are distinct and are separated by approximately 7.5 A, several substitutions of residues that interact with the bound RNA restored tryptophan binding to T30V TRAP. These observations support the hypothesis that conformational changes in TRAP relay information between the tryptophan and RNA binding sites of the protein.