Zinc Is Required for Assembly and Function of the Anti-trp RNA-binding Attenuation Protein, AT

Angela Valbuzzi,Charles Yanofsky

doi:10.1074/jbc.m208980200

Abstract

The anti-TRAP protein (AT) of Bacillus subtilis regulates expression of the trp operon and other genes concerned with tryptophan metabolism. AT acts by inhibiting the tryptophan-activated trp RNA-binding attenuation protein (TRAP). AT is an oligomer of identical 53-residue polypeptides; it is produced in response to the accumulation of uncharged tRNA(Trp). Each AT polypeptide has two cysteine-rich clusters that correspond to the signature motif of the cysteine-rich zinc-binding domain of the chaperone protein DnaJ. Here we characterize the putative zinc-binding domain of AT and establish the importance of zinc for AT assembly and activity. AT is shown to contain Zn(II) at a ratio of one ion per monomer. Bound zinc is necessary for maintenance of the quaternary structure of AT; the removal of zinc converts the AT complex into inactive monomers. All four cysteine residues in the AT polypeptide are involved in Zn(II) coordination. Chemical cross-linking analyses indicate that the AT functional oligomer is a hexamer composed of two trimers. Substituting alanine for any cysteine residue of AT results in rapid degradation of the mutant protein in vivo. We propose a model for the AT trimer in which three AT chains are held together by three zinc atoms, each coordinated by the N-terminal segment and the C-terminal segment of separate AT polypeptides.

Highlights

In Bacillus subtilis the genes of tryptophan metabolism are coordinately regulated in response to the cellular level of free tryptophan and the extent of charging of tRNATrp [1]
We examined anti-trp RNA-binding attenuation protein (TRAP) protein (AT) subunit association in native AT, HMB-AT, zinc-free AT, and zinc-reconstituted AT using cross-linking with glutaraldehyde (Fig. 1)
When the HMB reaction was reversed in the presence of the labilized zinc, reconstituted AT (ReAT), the protein appeared to return to its original structure (Fig. 1, lane 3)

Summary

EXPERIMENTAL PROCEDURES

Preparation of Zinc-free AT—Purified native AT was prepared as described previously [17]. The HMB reaction was reversed by incubating the recovered protein with the same Tris buffer containing 2 mM DTT plus 2 mM EDTA for 10 min at room temperature This zinc-free AT preparation was concentrated by centrifugation in a fresh Microcon device, and the concentrate was diluted with metal-free water to lower the DTT concentration to 0.5 mM. Zinc-reconstituted AT (ReAT) was prepared as described for zinc-free AT, except that EDTA was omitted, no wash steps were performed to remove the displaced zinc, and instead the sample was treated with DTT immediately following the last incubation with HMB (no exogenous zinc was added). The two amplified products corresponding to the 5Ј and 3Ј portions of the rtpA gene and partially overlapping in the central mutagenized region were gel-purified They were mixed, annealed, and used as template for the second step of PCR performed with the two external oligonucleotides. Bound antibody was visualized by use of horseradish peroxidaseconjugated donkey anti-rabbit immunoglobulin (Amersham Biosciences) and SuperSignal West Pico chemiluminescent detection reagents (Pierce)

RESULTS

ReATg ReAT

DISCUSSION

Angela Valbuzzi and Charles Yanofsky