The mechanism of tryptophan induction of tryptophanase operon expression: tryptophan inhibits release factor-mediated cleavage of TnaC-peptidyl-tRNA(Pro).
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.
Gong
F
F
Department of Biological Sciences, Stanford University, Stanford, CA 94305; and Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedal, Minato-Ku, Tokyo 108-8639, Japan.
Ito
K
K
Nakamura
Y
Y
Yanofsky
C
C
D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins; D006868:Hydrolysis; D009876:Operon; D011691:Puromycin / Q000037:antagonists & inhibitors; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism*; D012361:RNA, Transfer, Pro / Q000378:metabolism*; D014364:Tryptophan / Q000502:physiology; D014368:Tryptophanase / Q000235:genetics*
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D012346:RNA, Transfer, Amino Acyl; D012361:RNA, Transfer, Pro; C019037:tRNA, peptidyl-; C081473:tnaC protein, E coli; D011691:Puromycin; D014364:Tryptophan; D014368:Tryptophanase