Ribosomal features essential for tna operon induction: tryptophan binding at the peptidyl transferase center.
Features of the amino acid sequence of the TnaC nascent peptide are recognized by the translating ribosome. Recognition leads to tryptophan binding within the translating ribosome, inhibiting the termination of tnaC translation and preventing Rho-dependent transcription termination in the tna operon leader region. It was previously shown that inserting an adenine residue at position 751 or introducing the U2609C change in 23S rRNA or introducing the K90W replacement in ribosomal protein L22 prevented tryptophan induction of tna operon expression. It was also observed that an adenine at position 752 of 23S rRNA was required for induction. In the current study, the explanation for the lack of induction by these altered ribosomes was investigated. Using isolated TnaC-ribosome complexes, it was shown that although tryptophan inhibits puromycin cleavage of TnaC-tRNA(Pro) with wild-type ribosome complexes, it does not inhibit cleavage with the four mutant ribosome complexes examined. Similarly, tryptophan prevents sparsomycin inhibition of TnaC-tRNA(Pro) cleavage with wild-type ribosome complexes but not with these mutant ribosome complexes. Additionally, a nucleotide located close to the peptidyl transferase center, A2572, which was protected from methylation by tryptophan with wild-type ribosome complexes, was not protected with mutant ribosome complexes. These findings identify specific ribosomal residues located in the ribosome exit tunnel that recognize features of the TnaC peptide. This recognition creates a free tryptophan-binding site in the peptidyl transferase center, where bound tryptophan inhibits peptidyl transferase activity.
Cruz-Vera
Luis R
LR
Department of Biological Sciences, Stanford University, Stanford, CA 94305, USA.
New
Aaron
A
Squires
Catherine
C
Yanofsky
Charles
C
D001665:Binding Sites / Q000502:physiology; D048168:Escherichia coli K12 / Q000235:genetics* / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D008958:Models, Molecular; D008970:Molecular Weight; D009876:Operon / Q000235:genetics*; D010458:Peptidyl Transferases / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding / Q000502:physiology; D021122:Protein Subunits / Q000737:chemistry; D011691:Puromycin / Q000378:metabolism; D012269:Ribosomal Proteins / Q000096:biosynthesis* / Q000737:chemistry; D012270:Ribosomes / Q000737:chemistry / Q000378:metabolism*; D013033:Sparsomycin / Q000378:metabolism; D014364:Tryptophan / Q000378:metabolism*
D029968:Escherichia coli Proteins; D021122:Protein Subunits; D012269:Ribosomal Proteins; C081473:tnaC protein, E coli; D011691:Puromycin; D013033:Sparsomycin; D014364:Tryptophan; D010458:Peptidyl Transferases