Limits...
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.

Show MeSH

Related in: MedlinePlus

23S rRNA nucleotides that are protected by the TnaC nascent peptide. (A, C and E) Methylation protection assays were performed with ribosomes containing the indicated 23S rRNA alleles. Ribosomes translating (+) or not (−) messengers containing the tnaC gene sequences were analyzed in a buffer containing (+) or not (−) Trp. The ribosomes were exposed (+) or not (−) to the indicated alkylating agents. These assays were performed with [32P]-labeled oligonucleotides complementary to nucleotides 2654–2674 of 23S rRNA for (A and C), and complementary to nucleotides 821–838 of 23S rRNA for (E). Results obtained with ribosomes translating tnaC sequences in absences of the alkylating agents are shown in Supplementary Figure S3. Nucleotides methylated are indicated. cDNA synthesis on 23S rRNA template obtained from wild-type ribosomes is usually stopped by the naturally methylated G745 nucleotide (MG745) (E) (34). (B, D and F) Northern blot assays performed with the ribosomes indicated above. The ribosomal components were resolved in 10% denaturing Tris–tricine polyacrylamide gels. The presence of the TnaC-tRNAPro in the complexes was determined using a [32P]-labeled oligonucleotide complementary to the anti-codon region of the tRNAPro1 (35).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3299997&req=5

gkr1052-F2: 23S rRNA nucleotides that are protected by the TnaC nascent peptide. (A, C and E) Methylation protection assays were performed with ribosomes containing the indicated 23S rRNA alleles. Ribosomes translating (+) or not (−) messengers containing the tnaC gene sequences were analyzed in a buffer containing (+) or not (−) Trp. The ribosomes were exposed (+) or not (−) to the indicated alkylating agents. These assays were performed with [32P]-labeled oligonucleotides complementary to nucleotides 2654–2674 of 23S rRNA for (A and C), and complementary to nucleotides 821–838 of 23S rRNA for (E). Results obtained with ribosomes translating tnaC sequences in absences of the alkylating agents are shown in Supplementary Figure S3. Nucleotides methylated are indicated. cDNA synthesis on 23S rRNA template obtained from wild-type ribosomes is usually stopped by the naturally methylated G745 nucleotide (MG745) (E) (34). (B, D and F) Northern blot assays performed with the ribosomes indicated above. The ribosomal components were resolved in 10% denaturing Tris–tricine polyacrylamide gels. The presence of the TnaC-tRNAPro in the complexes was determined using a [32P]-labeled oligonucleotide complementary to the anti-codon region of the tRNAPro1 (35).

Mentions: The action of Trp on tna operon expression requires the presence of the TnaC nascent peptide within the ribosome (5). We performed methylation protection assays to determine if the nascent TnaC peptide protects 23S rRNA nucleotides A751, A752 and U2609 from chemical methylation (see ‘Materials and Methods’ section). These nucleotide residues are water accessible in vacant ribosomes (ribosomes not engaged in polypeptide synthesis), and can therefore be methylated by alkylating agents (41). We also examine methylation of U2585, which forms part of the PTC (25). The alkylating agent CMCT-induced methylation of U2585 and U2609 in vacant wild-type and +A751ins mutant ribosomes (Figure 2A, compare lane 2 with lane 1, or lane 4 with lane 3). We observed that the U2585 methylation level was slightly less (30 ± 5%, n = 4) in vacant +A751ins ribosomes than vacant wild-type ribosomes (Figure 2A, compare lane 4 with lane 2). We also observed that the methylation level of U2609 was slightly higher (25 ± 3%, n = 4) in vacant +A751ins ribosomes than in wild-type ribosomes (Figure 2A, compare lane 4 with lane 2). The A752C mutant ribosomes, like the +A751ins ribosomes, differed from the wild-type. Vacant A752C ribosomes showed slightly reduced methylation (32 ± 5%, n = 4) of U2585 and an increased methylation (50 ± 3%, n = 4) of U2609 compared to wild-type ribosomes (Figure 2C, compare lane 4 with lane 2). Trp did not affect methylation levels in vacant ribosomes (Supplementary Figure S2). These observations suggested that there were differences in the architecture of the ribosome exit tunnel between vacant +A751ins or A752C mutant ribosomes and wild-type ribosomes.Figure 2.


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)

23S rRNA nucleotides that are protected by the TnaC nascent peptide. (A, C and E) Methylation protection assays were performed with ribosomes containing the indicated 23S rRNA alleles. Ribosomes translating (+) or not (−) messengers containing the tnaC gene sequences were analyzed in a buffer containing (+) or not (−) Trp. The ribosomes were exposed (+) or not (−) to the indicated alkylating agents. These assays were performed with [32P]-labeled oligonucleotides complementary to nucleotides 2654–2674 of 23S rRNA for (A and C), and complementary to nucleotides 821–838 of 23S rRNA for (E). Results obtained with ribosomes translating tnaC sequences in absences of the alkylating agents are shown in Supplementary Figure S3. Nucleotides methylated are indicated. cDNA synthesis on 23S rRNA template obtained from wild-type ribosomes is usually stopped by the naturally methylated G745 nucleotide (MG745) (E) (34). (B, D and F) Northern blot assays performed with the ribosomes indicated above. The ribosomal components were resolved in 10% denaturing Tris–tricine polyacrylamide gels. The presence of the TnaC-tRNAPro in the complexes was determined using a [32P]-labeled oligonucleotide complementary to the anti-codon region of the tRNAPro1 (35).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkr1052-F2: 23S rRNA nucleotides that are protected by the TnaC nascent peptide. (A, C and E) Methylation protection assays were performed with ribosomes containing the indicated 23S rRNA alleles. Ribosomes translating (+) or not (−) messengers containing the tnaC gene sequences were analyzed in a buffer containing (+) or not (−) Trp. The ribosomes were exposed (+) or not (−) to the indicated alkylating agents. These assays were performed with [32P]-labeled oligonucleotides complementary to nucleotides 2654–2674 of 23S rRNA for (A and C), and complementary to nucleotides 821–838 of 23S rRNA for (E). Results obtained with ribosomes translating tnaC sequences in absences of the alkylating agents are shown in Supplementary Figure S3. Nucleotides methylated are indicated. cDNA synthesis on 23S rRNA template obtained from wild-type ribosomes is usually stopped by the naturally methylated G745 nucleotide (MG745) (E) (34). (B, D and F) Northern blot assays performed with the ribosomes indicated above. The ribosomal components were resolved in 10% denaturing Tris–tricine polyacrylamide gels. The presence of the TnaC-tRNAPro in the complexes was determined using a [32P]-labeled oligonucleotide complementary to the anti-codon region of the tRNAPro1 (35).
Mentions: The action of Trp on tna operon expression requires the presence of the TnaC nascent peptide within the ribosome (5). We performed methylation protection assays to determine if the nascent TnaC peptide protects 23S rRNA nucleotides A751, A752 and U2609 from chemical methylation (see ‘Materials and Methods’ section). These nucleotide residues are water accessible in vacant ribosomes (ribosomes not engaged in polypeptide synthesis), and can therefore be methylated by alkylating agents (41). We also examine methylation of U2585, which forms part of the PTC (25). The alkylating agent CMCT-induced methylation of U2585 and U2609 in vacant wild-type and +A751ins mutant ribosomes (Figure 2A, compare lane 2 with lane 1, or lane 4 with lane 3). We observed that the U2585 methylation level was slightly less (30 ± 5%, n = 4) in vacant +A751ins ribosomes than vacant wild-type ribosomes (Figure 2A, compare lane 4 with lane 2). We also observed that the methylation level of U2609 was slightly higher (25 ± 3%, n = 4) in vacant +A751ins ribosomes than in wild-type ribosomes (Figure 2A, compare lane 4 with lane 2). The A752C mutant ribosomes, like the +A751ins ribosomes, differed from the wild-type. Vacant A752C ribosomes showed slightly reduced methylation (32 ± 5%, n = 4) of U2585 and an increased methylation (50 ± 3%, n = 4) of U2609 compared to wild-type ribosomes (Figure 2C, compare lane 4 with lane 2). Trp did not affect methylation levels in vacant ribosomes (Supplementary Figure S2). These observations suggested that there were differences in the architecture of the ribosome exit tunnel between vacant +A751ins or A752C mutant ribosomes and wild-type ribosomes.Figure 2.

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