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PMID: 16505360 Published · ppublish English Journal Article Research Support, U.S. Gov't, Non-P.H.S.

Changes produced by bound tryptophan in the ribosome peptidyl transferase center in response to TnaC, a nascent leader peptide.

Proceedings of the National Academy of Sciences of the United States of America ·Vol. 103 ·No. 10 ·2006-03-07 ·Pages 3598-603

Cruz-Vera LR, Gong M, Yanofsky C

Abstract

Studies in vitro have established that free tryptophan induces tna operon expression by binding to the ribosome that has just completed synthesis of TnaC-tRNA(Pro), the peptidyl-tRNA precursor of the leader peptide of this operon. Tryptophan acts by inhibiting Release Factor 2-mediated cleavage of this peptidyl-tRNA at the tnaC stop codon. Here we analyze the ribosomal location of free tryptophan, the changes it produces in the ribosome, and the role of the nascent TnaC-tRNA(Pro) peptide in facilitating tryptophan binding and induction. The positional changes of 23S rRNA nucleotides that occur during induction were detected by using methylation protection and binding/competition assays. The ribosome-TnaC-tRNA(Pro) complexes analyzed were formed in vitro; they contained either wild-type TnaC-tRNA(Pro) or its nonfunctional substitute, TnaC(W12R)-tRNA(Pro). Upon comparing these two peptidyl-tRNA-ribosome complexes, free tryptophan was found to block methylation of nucleotide A2572 of wild-type ribosome-TnaC-tRNA(Pro) complexes but not of ribosome-TnaC(W12R)-tRNA(Pro) complexes. Nucleotide A2572 is in the ribosomal peptidyl transferase center. Tryptophanol, a noninducing competitor of tryptophan, was ineffective in blocking A2572 methylation; however, it did reverse the protective effect of tryptophan. Free tryptophan inhibited puromycin cleavage of TnaC-tRNA(Pro); it also inhibited binding of the antibiotic sparsomycin. These effects were not observed with TnaC(W12R)-tRNA(Pro) mutant complexes. These findings establish that Trp-12 of TnaC-tRNA(Pro) is required for introducing specific changes in the peptidyl transferase center of the ribosome that activate free tryptophan binding, resulting in peptidyl transferase inhibition. Free tryptophan appears to act at or near the binding sites of several antibiotics in the peptidyl transferase center.

MeSH Terms
Amino Acid Substitution Base Sequence Binding Sites/genetics Escherichia coli/genetics,metabolism Escherichia coli Proteins/chemistry,genetics,metabolism Macromolecular Substances Methylation Models, Molecular Operon Peptidyl Transferases/chemistry,genetics,metabolism Puromycin/metabolism RNA, Bacterial/chemistry,genetics,metabolism RNA, Transfer, Amino Acyl/metabolism RNA, Transfer, Pro/chemistry,genetics,metabolism Recombinant Proteins/chemistry,genetics,metabolism Ribosomes/enzymology Tryptophan/chemistry
Chemicals
Escherichia coli Proteins Macromolecular Substances RNA, Bacterial RNA, Transfer, Amino Acyl RNA, Transfer, Pro Recombinant Proteins tnaC protein, E coli Puromycin Tryptophan Peptidyl Transferases
Authors & Affiliations
3 authors, click to expand affiliations / ORCID
Cruz-Vera Luis Rogelio
Department of Biological Sciences, Stanford University, Stanford, CA 94305-5020, USA.
Gong Ming
Yanofsky Charles
References (32)
32 references, click to expand
  1. In vitro translation of the upstream open reading frame in the mammalian mRNA encoding S-adenosylmethionine decarboxylase.
    J Biol Chem. 2000 Aug 11;275(32):24444-50 PMID: 10829027
  2. Overexpression of tnaC of Escherichia coli inhibits growth by depleting tRNA2Pro availability.
    J Bacteriol. 2006 Mar;188(5):1892-8 PMID: 16484200
  3. Upstream open reading frames as regulators of mRNA translation.
    Mol Cell Biol. 2000 Dec;20(23):8635-42 PMID: 11073965
  4. The mechanism of tryptophan induction of tryptophanase operon expression: tryptophan inhibits release factor-mediated cleavage of TnaC-peptidyl-tRNA(Pro).
    Proc Natl Acad Sci U S A. 2001 Jul 31;98(16):8997-9001 PMID: 11470925
  5. A conformational change in the ribosomal peptidyl transferase center upon active/inactive transition.
    Proc Natl Acad Sci U S A. 2001 Aug 28;98(18):10096-101 PMID: 11517305
  6. Regulatory nascent peptides in the ribosomal tunnel.
    Cell. 2002 Mar 8;108(5):591-4 PMID: 11893330
  7. The ribosomal exit tunnel functions as a discriminating gate.
    Cell. 2002 Mar 8;108(5):629-36 PMID: 11893334
  8. Analysis of tryptophanase operon expression in vitro: accumulation of TnaC-peptidyl-tRNA in a release factor 2-depleted S-30 extract prevents Rho factor action, simulating induction.
    J Biol Chem. 2002 May 10;277(19):17095-100 PMID: 11880383
  9. Instruction of translating ribosome by nascent peptide.
    Science. 2002 Sep 13;297(5588):1864-7 PMID: 12228716
  10. A nascent polypeptide domain that can regulate translation elongation.
    Proc Natl Acad Sci U S A. 2004 Mar 23;101(12):4059-64 PMID: 15020769
  11. The active site of the ribosome is composed of two layers of conserved nucleotides with distinct roles in peptide bond formation and peptide release.
    Cell. 2004 May 28;117(5):589-99 PMID: 15163407
  12. Nucleotide sequence of the structural gene for tryptophanase of Escherichia coli K-12.
    J Bacteriol. 1981 Sep;147(3):787-96 PMID: 6268608
  13. Transcription initiation at the tryptophanase promoter of Escherichia coli K-12.
    J Bacteriol. 1982 Aug;151(2):942-51 PMID: 6284718
  14. Rho-dependent transcription termination in the tryptophanase operon leader region of Escherichia coli K-12.
    J Bacteriol. 1986 Apr;166(1):217-23 PMID: 2420781
  15. Structural analysis of RNA using chemical and enzymatic probing monitored by primer extension.
    Methods Enzymol. 1988;164:481-9 PMID: 2468070
  16. tRNA(Trp) translation of leader peptide codon 12 and other factors that regulate expression of the tryptophanase operon.
    J Bacteriol. 1990 Jun;172(6):3100-7 PMID: 2345136
  17. Direct analysis of aminoacylation levels of tRNAs in vivo. Application to studying recognition of Escherichia coli initiator tRNA mutants by glutaminyl-tRNA synthetase.
    J Biol Chem. 1991 Dec 25;266(36):24712-8 PMID: 1761566
  18. Mapping the rRNA neighborhood of the acceptor end of tRNA in the ribosome.
    EMBO J. 1996 Feb 15;15(4):910-6 PMID: 8631311
  19. Ribosome regulation by the nascent peptide.
    Microbiol Rev. 1996 Jun;60(2):366-85 PMID: 8801438
  20. Co-variation of tRNA abundance and codon usage in Escherichia coli at different growth rates.
    J Mol Biol. 1996 Aug 2;260(5):649-63 PMID: 8709146
  21. Mutations in the peptidyl transferase center of 23 S rRNA reveal the site of action of sparsomycin, a universal inhibitor of translation.
    J Mol Biol. 1996 Aug 16;261(2):222-30 PMID: 8757289
  22. A sparsomycin-resistant mutant of Halobacterium salinarium lacks a modification at nucleotide U2603 in the peptidyl transferase centre of 23 S rRNA.
    J Mol Biol. 1996 Aug 16;261(2):231-8 PMID: 8757290
  23. Inhibition of nascent-peptide release at translation termination.
    Mol Cell Biol. 1996 Dec;16(12):7109-14 PMID: 8943366
  24. Regulation of the Escherichia coli tna operon: nascent leader peptide control at the tnaC stop codon.
    J Bacteriol. 1997 Mar;179(5):1774-9 PMID: 9045840
  25. Ribosome-catalyzed peptide-bond formation with an A-site substrate covalently linked to 23S ribosomal RNA.
    Science. 1998 Apr 10;280(5361):286-9 PMID: 9535658
  26. Direct crosslinking of the antitumor antibiotic sparsomycin, and its derivatives, to A2602 in the peptidyl transferase center of 23S-like rRNA within ribosome-tRNA complexes.
    Proc Natl Acad Sci U S A. 1999 Aug 3;96(16):9003-8 PMID: 10430885
  27. Mutational analysis of 23S ribosomal RNA structure and function in Escherichia coli.
    Adv Genet. 1999;41:157-95 PMID: 10494619
  28. Acetylornithinase of Escherichia coli: partial purification and some properties.
    J Biol Chem. 1956 Jan;218(1):97-106 PMID: 13278318
  29. Features of ribosome-peptidyl-tRNA interactions essential for tryptophan induction of tna operon expression.
    Mol Cell. 2005 Aug 5;19(3):333-43 PMID: 16061180
  30. Essential mechanisms in the catalysis of peptide bond formation on the ribosome.
    J Biol Chem. 2005 Oct 28;280(43):36065-72 PMID: 16129670
  31. Structures of the bacterial ribosome at 3.5 A resolution.
    Science. 2005 Nov 4;310(5749):827-34 PMID: 16272117
  32. The complete atomic structure of the large ribosomal subunit at 2.4 A resolution.
    Science. 2000 Aug 11;289(5481):905-20 PMID: 10937989
Article Info
Journal
Proceedings of the National Academy of Sciences of the United States of America
Abbr.
Proc Natl Acad Sci U S A
ISSN
0027-8424
DOI
10.1073/pnas.0600082103
Published
2006-03-07
Epub
2006-00-27
Pages
3598-603
Language
English
Region
United States
NLM ID
7505876
PMCID
PMC1450129
Subset
IM
External Links
PubMed source DOI: 10.1073/pnas.0600082103 PMC Full Text View journal details
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