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Crucial elements that maintain the interactions between the regulatory TnaC peptide and the ribosome exit tunnel responsible for Trp inhibition of ribosome function.

Martínez AK, Shirole NH, Murakami S, Benedik MJ, Sachs MS, Cruz-Vera LR - Nucleic Acids Res. (2011)

Bottom Line: Nucleotides A752 and U2609 establish a base-pair interaction.These data indicate that the TnaC nascent peptide in the ribosome exit tunnel interacts with the U2609 nucleotide when the ribosome is Trp responsive.This interaction is affected by mutational changes in exit tunnel nucleotides of 23S rRNA, as well as in conserved TnaC residues, suggesting that they affect the structure of the exit tunnel and/or the nascent peptide configuration in the tunnel.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Texas A&M University, College Station, TX 77843, USA.

ABSTRACT
Translation of the TnaC nascent peptide inhibits ribosomal activity in the presence of l-tryptophan, inducing expression of the tnaCAB operon in Escherichia coli. Using chemical methylation, this work reveals how interactions between TnaC and the ribosome are affected by mutations in both molecules. The presence of the TnaC-tRNA(Pro) peptidyl-tRNA within the ribosome protects the 23S rRNA nucleotide U2609 against chemical methylation. Such protection was not observed in mutant ribosomes containing changes in 23S rRNA nucleotides of the A748-A752 region. Nucleotides A752 and U2609 establish a base-pair interaction. Most replacements of either A752 or U2609 affected Trp induction of a TnaC-regulated LacZ reporter. However, the single change A752G, or the dual replacements A752G and U2609C, maintained Trp induction. Replacements at the conserved TnaC residues W12 and D16 also abolished the protection of U2609 by TnaC-tRNA(Pro) against chemical methylation. These data indicate that the TnaC nascent peptide in the ribosome exit tunnel interacts with the U2609 nucleotide when the ribosome is Trp responsive. This interaction is affected by mutational changes in exit tunnel nucleotides of 23S rRNA, as well as in conserved TnaC residues, suggesting that they affect the structure of the exit tunnel and/or the nascent peptide configuration in the tunnel.

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Mutations of 23S rRNA nucleotides that affect tna operon expression. Bacterial cells expressing the indicated 23S rRNA alleles were used to analyze expression of β-gal from a tnaC-tnaA’-‘lacZ protein fusion. Bacterial cultures were grown in minimal medium containing 0.2% glycerol, 0.05% acid-hydrolyzed casein, 0.01% vitamin B1 and variable amounts of 1 MT as an inducer. The figure on the right shows an amplification of the plots between the induction values zero to four.
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gkr1052-F1: Mutations of 23S rRNA nucleotides that affect tna operon expression. Bacterial cells expressing the indicated 23S rRNA alleles were used to analyze expression of β-gal from a tnaC-tnaA’-‘lacZ protein fusion. Bacterial cultures were grown in minimal medium containing 0.2% glycerol, 0.05% acid-hydrolyzed casein, 0.01% vitamin B1 and variable amounts of 1 MT as an inducer. The figure on the right shows an amplification of the plots between the induction values zero to four.

Mentions: Mutations in 23S rRNA nucleotides that constitute the ribosome exit tunnel reduce TnaC-mediated operon induction in response to Trp (19,22). We used bacterial strains in which the seven rRNA operons were deleted from their chromosomal locations to analyze how A752C, U2609C and +A751 insertion mutations in the 23S rRNA affected the inhibition of ribosome function by Trp (19). These strains contained a homogeneous population of mutant ribosomes (Supplementary Figure S1). To determine the effect of ribosomal mutations on TnaC-mediated regulation in response to Trp and Trp analogs, we used bacteria that contained a tnaC tnaA’-‘lacZ reporter gene (19). We tested the effects of these ribosomal mutations on the expression of the reporter gene in vivo using several concentrations of 1-methyl-l-Trp (1MT). This Trp analog induces operon expression but is not cleaved by tryptophanase and thus is a more efficient inducer in vivo than Trp (42). The results are summarized in Figure 1 (primary data are given in Supplementary Table S1). Based on the induction curve, 40 µM of 1MT would be sufficient for maximal expression of the reporter gene in cells containing wild-type ribosomes (Figure 1, closed circles). For cells containing ribosomes with the +A751ins mutation, even at 100 µM 1MT operon induction was 12-fold lower than cells containing wild-type ribosomes (Figure 1, compare open squares with closed circles). Similar results were observed with cells containing A752C mutant ribosomes, in which 100 µM 1MT induced 25-fold less operon expression than in wild-type cells (Figure 1, compare open triangles with closed circles). These results indicated that +A751ins or A752C ribosomes allowed at most, slight induction of the tna reporter operon at high concentrations of 1MT (Figure 1, inset). Finally, we observed in cells containing ribosomes with the U2609C replacement that expression of the reporter gene was not induced by the addition of any amount of 1MT tested (Figure 1, open circles and data not shown). This result indicated that the U2609C replacement completely abolished TnaC-mediated regulation in response to 1MT. In summary, we observed differences in the way that the +A751ins, A752C and the U2609C mutations affected TnaC-mediated regulation.Figure 1.


Crucial elements that maintain the interactions between the regulatory TnaC peptide and the ribosome exit tunnel responsible for Trp inhibition of ribosome function.

Martínez AK, Shirole NH, Murakami S, Benedik MJ, Sachs MS, Cruz-Vera LR - Nucleic Acids Res. (2011)

Mutations of 23S rRNA nucleotides that affect tna operon expression. Bacterial cells expressing the indicated 23S rRNA alleles were used to analyze expression of β-gal from a tnaC-tnaA’-‘lacZ protein fusion. Bacterial cultures were grown in minimal medium containing 0.2% glycerol, 0.05% acid-hydrolyzed casein, 0.01% vitamin B1 and variable amounts of 1 MT as an inducer. The figure on the right shows an amplification of the plots between the induction values zero to four.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3299997&req=5

gkr1052-F1: Mutations of 23S rRNA nucleotides that affect tna operon expression. Bacterial cells expressing the indicated 23S rRNA alleles were used to analyze expression of β-gal from a tnaC-tnaA’-‘lacZ protein fusion. Bacterial cultures were grown in minimal medium containing 0.2% glycerol, 0.05% acid-hydrolyzed casein, 0.01% vitamin B1 and variable amounts of 1 MT as an inducer. The figure on the right shows an amplification of the plots between the induction values zero to four.
Mentions: Mutations in 23S rRNA nucleotides that constitute the ribosome exit tunnel reduce TnaC-mediated operon induction in response to Trp (19,22). We used bacterial strains in which the seven rRNA operons were deleted from their chromosomal locations to analyze how A752C, U2609C and +A751 insertion mutations in the 23S rRNA affected the inhibition of ribosome function by Trp (19). These strains contained a homogeneous population of mutant ribosomes (Supplementary Figure S1). To determine the effect of ribosomal mutations on TnaC-mediated regulation in response to Trp and Trp analogs, we used bacteria that contained a tnaC tnaA’-‘lacZ reporter gene (19). We tested the effects of these ribosomal mutations on the expression of the reporter gene in vivo using several concentrations of 1-methyl-l-Trp (1MT). This Trp analog induces operon expression but is not cleaved by tryptophanase and thus is a more efficient inducer in vivo than Trp (42). The results are summarized in Figure 1 (primary data are given in Supplementary Table S1). Based on the induction curve, 40 µM of 1MT would be sufficient for maximal expression of the reporter gene in cells containing wild-type ribosomes (Figure 1, closed circles). For cells containing ribosomes with the +A751ins mutation, even at 100 µM 1MT operon induction was 12-fold lower than cells containing wild-type ribosomes (Figure 1, compare open squares with closed circles). Similar results were observed with cells containing A752C mutant ribosomes, in which 100 µM 1MT induced 25-fold less operon expression than in wild-type cells (Figure 1, compare open triangles with closed circles). These results indicated that +A751ins or A752C ribosomes allowed at most, slight induction of the tna reporter operon at high concentrations of 1MT (Figure 1, inset). Finally, we observed in cells containing ribosomes with the U2609C replacement that expression of the reporter gene was not induced by the addition of any amount of 1MT tested (Figure 1, open circles and data not shown). This result indicated that the U2609C replacement completely abolished TnaC-mediated regulation in response to 1MT. In summary, we observed differences in the way that the +A751ins, A752C and the U2609C mutations affected TnaC-mediated regulation.Figure 1.

Bottom Line: Nucleotides A752 and U2609 establish a base-pair interaction.These data indicate that the TnaC nascent peptide in the ribosome exit tunnel interacts with the U2609 nucleotide when the ribosome is Trp responsive.This interaction is affected by mutational changes in exit tunnel nucleotides of 23S rRNA, as well as in conserved TnaC residues, suggesting that they affect the structure of the exit tunnel and/or the nascent peptide configuration in the tunnel.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Texas A&M University, College Station, TX 77843, USA.

ABSTRACT
Translation of the TnaC nascent peptide inhibits ribosomal activity in the presence of l-tryptophan, inducing expression of the tnaCAB operon in Escherichia coli. Using chemical methylation, this work reveals how interactions between TnaC and the ribosome are affected by mutations in both molecules. The presence of the TnaC-tRNA(Pro) peptidyl-tRNA within the ribosome protects the 23S rRNA nucleotide U2609 against chemical methylation. Such protection was not observed in mutant ribosomes containing changes in 23S rRNA nucleotides of the A748-A752 region. Nucleotides A752 and U2609 establish a base-pair interaction. Most replacements of either A752 or U2609 affected Trp induction of a TnaC-regulated LacZ reporter. However, the single change A752G, or the dual replacements A752G and U2609C, maintained Trp induction. Replacements at the conserved TnaC residues W12 and D16 also abolished the protection of U2609 by TnaC-tRNA(Pro) against chemical methylation. These data indicate that the TnaC nascent peptide in the ribosome exit tunnel interacts with the U2609 nucleotide when the ribosome is Trp responsive. This interaction is affected by mutational changes in exit tunnel nucleotides of 23S rRNA, as well as in conserved TnaC residues, suggesting that they affect the structure of the exit tunnel and/or the nascent peptide configuration in the tunnel.

Show MeSH
Related in: MedlinePlus