The mechanism of tryptophan induction of tryptophanase operon expression: tryptophan inhibits release factor-mediated cleavage of TnaC-peptidyl-tRNA(Pro)

Abstract

Expression of the tryptophanase (tna) operon of Escherichia coli is regulated by catabolite repression and tryptophan-induced transcription antitermination. In a previous study, we reproduced the regulatory features of this operon observed in vivo by using an in vitro S-30 system. We also found that, under inducing conditions, the leader peptidyl-tRNA (TnaC-peptidyl-tRNA(Pro)) is not cleaved; it accumulates in the S-30 reaction mixture. In this paper, we examine the requirements for TnaC-peptidyl-tRNA(Pro) accumulation and cleavage, in vitro. We show that this peptidyl-tRNA remains bound to the translating ribosome. Removal of free tryptophan and addition of release factor 1 or 2 leads to hydrolysis of TnaC-peptidyl-tRNA(Pro) and release of TnaC from the ribosome-mRNA complex. Release factor-mediated cleavage is prevented by the addition of tryptophan. TnaC of the ribosome-bound TnaC-peptidyl-tRNA(Pro) was transferable to puromycin. This transfer was also blocked by tryptophan. Tests with various tryptophan analogs as substitutes for tryptophan revealed the existence of strict structural requirements for tryptophan action. Our findings demonstrate that the addition of tryptophan to ribosomes bearing nascent TnaC-peptidyl-tRNA(Pro) inhibits both TnaC peptidyl-tRNA(Pro) hydrolysis and TnaC peptidyl transfer. The associated translating ribosome therefore remains attached to the leader transcript where it blocks Rho factor binding and subsequent transcription termination.

Figure 1

[³⁵S]TnaC peptidyl-tRNA^Pro is ribosome associated under inducing condition. A tryptophan-induced S-30 reaction mixture was centrifuged, and the ribosome pellet was resuspended in TMKN buffer. Portions of the ribosome pellet (P) and supernatant (S) were analyzed electrophoretically (Upper Inset). The remainder of the resuspended ribosome pellet was layered on a 10–30% sucrose gradient and centrifuged. Fractions were collected, and absorbance at 260 nm was monitored (Upper). Proteins in each fraction were recovered by cold acetone precipitation, dissolved in 20 μl 1× Tricine-SDS gel sample buffer, separated electrophoretically, and analyzed by autoradiography. Prestained protein molecular weight standards were used as markers (not shown); TnaC-tRNA^Pro was found to be ≈25 kDa (Lower).

Figure 2

Release factor-mediated cleavage of TnaC-tRNA^Pro. TnaC-tRNA^Pro (open arrow) and TnaC (filled arrow) bands are indicated. (A). Spontaneous cleavage of TnaC-tRNA^Pro is not evident on incubation of isolated ribosome complexes at 37°C. Samples were taken at the indicated times and analyzed by electrophoresis and autoradiography. (B) RF-2-mediated TnaC-tRNA^Pro cleavage and its inhibition by l-tryptophan. Ribosome complexes with the indicated additions were incubated at 37°C for 15 min, and then analyzed electrophoretically. The TnaC-tRNA^Pro concentration in lane 1 was set at 100%. (C) A comparison of RF1- and RF2-mediated TnaC-tRNA^Pro cleavage. Incubation was performed as in the middle figure. It was also found (not shown) that there is no significant cleavage when GTP is added with RF3. The TnaC-tRNA^Pro concentration in lane 1 was set at 100%.

Figure 3

Transfer of TnaC from isolated TnaC peptidyl-tRNA^Pro⋅tna mRNA⋅ribosome complexes to puromycin. TnaC, TnaC-puromycin (filled arrow), and TnaC-tRNA^Pro (open arrow) bands are marked. (A) Puromycin-mediated TnaC peptidyl transfer. Ribosome complexes were mixed with 1 mM l-tryptophan, indole, 1-methyl-l-tryptophan, or puromycin, respectively, and incubated at 37°C for 15 min. A sample containing puromycin was also incubated for 30 min. (B) Chloramphenicol (Cm) or tryptophan inhibits puromycin-mediated TnaC peptidyl transfer. Experimental details are described in Materials and Methods. The TnaC peptidyl-tRNA^Pro concentration was not changed when Cm alone was present (not shown). The TnaC-tRNA^Pro concentration in lane 1 was set at 100%. It is unclear why TnaC and TnaC-puromycin signals in lane 4 are stronger than those in lane 3 on this gel, but this discrepancy was not generally observed. No significant differences in the stability of TnaC and TnaC-puromycin were detected.

Figure 4

Analysis of the structural features of l-tryptophan required for inhibition of puromycin-mediated TnaC peptidyl transfer. TnaC (filled arrow) and TnaC-tRNA^Pro (open arrow) bands are indicated. Ribosome complexes with the indicated additions were incubated at 37°C for 15 min, and then analyzed electrophoretically. (A and B) Test of the ability of tryptophan analogs (2 mM) to inhibit puromycin-mediated TnaC peptidyl transfer. (C) Tryptamine prevents l-tryptophan inhibition of puromycin-mediated TnaC transfer. IPA, indole-3-propionic acid.