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Prevention of cross-talk in conserved regulatory systems: identification of specificity determinants in RNA-binding anti-termination proteins of the BglG family.

Hübner S, Declerck N, Diethmaier C, Le Coq D, Aymerich S, Stülke J - Nucleic Acids Res. (2011)

Bottom Line: This analysis revealed the key role of an arginine side-chain for both the high affinity and specificity of LicT for its cognate RAT.Introduction of this Arg at the equivalent position of SacY (A26) increased the RNA binding in vitro but also resulted in a relaxed specificity.Altogether our results suggest that this family of anti-termination proteins has evolved to reach a compromise between RNA binding efficacy and specific interaction with individual target sequences.

View Article: PubMed Central - PubMed

Affiliation: Department of General Microbiology, Institute of Microbiology and Genetics, Georg-August University Göttingen, Grisebach strasse 8, D-37077 Göttingen, Germany.

ABSTRACT
Each family of signal transduction systems requires specificity determinants that link individual signals to the correct regulatory output. In Bacillus subtilis, a family of four anti-terminator proteins controls the expression of genes for the utilisation of alternative sugars. These regulatory systems contain the anti-terminator proteins and a RNA structure, the RNA anti-terminator (RAT) that is bound by the anti-terminator proteins. We have studied three of these proteins (SacT, SacY, and LicT) to understand how they can transmit a specific signal in spite of their strong structural homology. A screen for random mutations that render SacT capable to bind a RNA structure recognized by LicT only revealed a substitution (P26S) at one of the few non-conserved residues that are in contact with the RNA. We have randomly modified this position in SacT together with another non-conserved RNA-contacting residue (Q31). Surprisingly, the mutant proteins could bind all RAT structures that are present in B. subtilis. In a complementary approach, reciprocal amino acid exchanges have been introduced in LicT and SacY at non-conserved positions of the RNA-binding site. This analysis revealed the key role of an arginine side-chain for both the high affinity and specificity of LicT for its cognate RAT. Introduction of this Arg at the equivalent position of SacY (A26) increased the RNA binding in vitro but also resulted in a relaxed specificity. Altogether our results suggest that this family of anti-termination proteins has evolved to reach a compromise between RNA binding efficacy and specific interaction with individual target sequences.

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Conservation of the regulatory components of the anti-termination systems of the BglG/ SacY family. (A) Summary of the relevant RAT structures. Boxes indicate nucleotides that differ from the cognate wild-type RAT. For licTopt RAT positions 3, 4 and 26 are mutated leading to a complete LicT dependent RAT structure. sacB was mutated that the RAT structure resembles sacP (sacB-R6) and bglP (sacB-R8) (18). These recombinant RAT structures are designated sacPR and bglPR, respectively. Circles indicate the 2 nt that are not conserved in the sacB RAT and licS RAT used in this study. The position of the asymmetric internal loops (1 and 2) characterizing the RAT hairpin is indicated. (B) Alignment of the CAT domains of the four anti-termination proteins of the BglG/SacY family in B. subtilis. Boxes indicate the amino acids involved in RNA recognition according to the structure of the LicT CAT/RAT complex (PDB ID code 1L1C) (17). Underlined residues in SacY CAT indicate the RNA-contacting region as mapped by NMR titration (5). Non-conserved amino acids that have been targeted for mutagenesis are labelled by arrows.
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Figure 1: Conservation of the regulatory components of the anti-termination systems of the BglG/ SacY family. (A) Summary of the relevant RAT structures. Boxes indicate nucleotides that differ from the cognate wild-type RAT. For licTopt RAT positions 3, 4 and 26 are mutated leading to a complete LicT dependent RAT structure. sacB was mutated that the RAT structure resembles sacP (sacB-R6) and bglP (sacB-R8) (18). These recombinant RAT structures are designated sacPR and bglPR, respectively. Circles indicate the 2 nt that are not conserved in the sacB RAT and licS RAT used in this study. The position of the asymmetric internal loops (1 and 2) characterizing the RAT hairpin is indicated. (B) Alignment of the CAT domains of the four anti-termination proteins of the BglG/SacY family in B. subtilis. Boxes indicate the amino acids involved in RNA recognition according to the structure of the LicT CAT/RAT complex (PDB ID code 1L1C) (17). Underlined residues in SacY CAT indicate the RNA-contacting region as mapped by NMR titration (5). Non-conserved amino acids that have been targeted for mutagenesis are labelled by arrows.

Mentions: In addition to LicT, the GlcT anti-terminator protein controls the expression of the ptsG gene encoding the glucose permease of the PTS, and SacT and SacY regulate the sacPA and sacB genes, respectively, that are involved in sucrose utilization [for a review see (6)]. As described for LicT, the cognate sugar-specific PTS permeases and HPr phosphorylate, these proteins thus control their activity (14). If properly phosphorylated, they bind to their respective RAT structures in the ptsG or sacPA and sacB mRNAs and cause transcriptional anti-termination. These four regulatory systems share multiple levels of similarity: (i) the anti-termination proteins are conserved, (ii) the PTS components that phosphorylate the PRD-I are similar to each other and (iii) the RAT structures recognized by the CATs of the four anti-terminator proteins also share extensive similarity (Figure 1). Thus, it is not surprising that cross-talk between the anti-termination proteins and non-cognate RAT structures was observed (15,16). The complex formed between the CAT of LicT and the bglPH RAT has been studied by NMR, and it turned out that LicT contacts bases in the two internal loops (or bulges) of the RAT. The basic stretch at the N-terminus of the CAT (residues 5–10) and the residues Gly-26, Arg-27, Phe-31 and Gln-32 are involved in these contacts (17).Figure 1.


Prevention of cross-talk in conserved regulatory systems: identification of specificity determinants in RNA-binding anti-termination proteins of the BglG family.

Hübner S, Declerck N, Diethmaier C, Le Coq D, Aymerich S, Stülke J - Nucleic Acids Res. (2011)

Conservation of the regulatory components of the anti-termination systems of the BglG/ SacY family. (A) Summary of the relevant RAT structures. Boxes indicate nucleotides that differ from the cognate wild-type RAT. For licTopt RAT positions 3, 4 and 26 are mutated leading to a complete LicT dependent RAT structure. sacB was mutated that the RAT structure resembles sacP (sacB-R6) and bglP (sacB-R8) (18). These recombinant RAT structures are designated sacPR and bglPR, respectively. Circles indicate the 2 nt that are not conserved in the sacB RAT and licS RAT used in this study. The position of the asymmetric internal loops (1 and 2) characterizing the RAT hairpin is indicated. (B) Alignment of the CAT domains of the four anti-termination proteins of the BglG/SacY family in B. subtilis. Boxes indicate the amino acids involved in RNA recognition according to the structure of the LicT CAT/RAT complex (PDB ID code 1L1C) (17). Underlined residues in SacY CAT indicate the RNA-contacting region as mapped by NMR titration (5). Non-conserved amino acids that have been targeted for mutagenesis are labelled by arrows.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
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Figure 1: Conservation of the regulatory components of the anti-termination systems of the BglG/ SacY family. (A) Summary of the relevant RAT structures. Boxes indicate nucleotides that differ from the cognate wild-type RAT. For licTopt RAT positions 3, 4 and 26 are mutated leading to a complete LicT dependent RAT structure. sacB was mutated that the RAT structure resembles sacP (sacB-R6) and bglP (sacB-R8) (18). These recombinant RAT structures are designated sacPR and bglPR, respectively. Circles indicate the 2 nt that are not conserved in the sacB RAT and licS RAT used in this study. The position of the asymmetric internal loops (1 and 2) characterizing the RAT hairpin is indicated. (B) Alignment of the CAT domains of the four anti-termination proteins of the BglG/SacY family in B. subtilis. Boxes indicate the amino acids involved in RNA recognition according to the structure of the LicT CAT/RAT complex (PDB ID code 1L1C) (17). Underlined residues in SacY CAT indicate the RNA-contacting region as mapped by NMR titration (5). Non-conserved amino acids that have been targeted for mutagenesis are labelled by arrows.
Mentions: In addition to LicT, the GlcT anti-terminator protein controls the expression of the ptsG gene encoding the glucose permease of the PTS, and SacT and SacY regulate the sacPA and sacB genes, respectively, that are involved in sucrose utilization [for a review see (6)]. As described for LicT, the cognate sugar-specific PTS permeases and HPr phosphorylate, these proteins thus control their activity (14). If properly phosphorylated, they bind to their respective RAT structures in the ptsG or sacPA and sacB mRNAs and cause transcriptional anti-termination. These four regulatory systems share multiple levels of similarity: (i) the anti-termination proteins are conserved, (ii) the PTS components that phosphorylate the PRD-I are similar to each other and (iii) the RAT structures recognized by the CATs of the four anti-terminator proteins also share extensive similarity (Figure 1). Thus, it is not surprising that cross-talk between the anti-termination proteins and non-cognate RAT structures was observed (15,16). The complex formed between the CAT of LicT and the bglPH RAT has been studied by NMR, and it turned out that LicT contacts bases in the two internal loops (or bulges) of the RAT. The basic stretch at the N-terminus of the CAT (residues 5–10) and the residues Gly-26, Arg-27, Phe-31 and Gln-32 are involved in these contacts (17).Figure 1.

Bottom Line: This analysis revealed the key role of an arginine side-chain for both the high affinity and specificity of LicT for its cognate RAT.Introduction of this Arg at the equivalent position of SacY (A26) increased the RNA binding in vitro but also resulted in a relaxed specificity.Altogether our results suggest that this family of anti-termination proteins has evolved to reach a compromise between RNA binding efficacy and specific interaction with individual target sequences.

View Article: PubMed Central - PubMed

Affiliation: Department of General Microbiology, Institute of Microbiology and Genetics, Georg-August University Göttingen, Grisebach strasse 8, D-37077 Göttingen, Germany.

ABSTRACT
Each family of signal transduction systems requires specificity determinants that link individual signals to the correct regulatory output. In Bacillus subtilis, a family of four anti-terminator proteins controls the expression of genes for the utilisation of alternative sugars. These regulatory systems contain the anti-terminator proteins and a RNA structure, the RNA anti-terminator (RAT) that is bound by the anti-terminator proteins. We have studied three of these proteins (SacT, SacY, and LicT) to understand how they can transmit a specific signal in spite of their strong structural homology. A screen for random mutations that render SacT capable to bind a RNA structure recognized by LicT only revealed a substitution (P26S) at one of the few non-conserved residues that are in contact with the RNA. We have randomly modified this position in SacT together with another non-conserved RNA-contacting residue (Q31). Surprisingly, the mutant proteins could bind all RAT structures that are present in B. subtilis. In a complementary approach, reciprocal amino acid exchanges have been introduced in LicT and SacY at non-conserved positions of the RNA-binding site. This analysis revealed the key role of an arginine side-chain for both the high affinity and specificity of LicT for its cognate RAT. Introduction of this Arg at the equivalent position of SacY (A26) increased the RNA binding in vitro but also resulted in a relaxed specificity. Altogether our results suggest that this family of anti-termination proteins has evolved to reach a compromise between RNA binding efficacy and specific interaction with individual target sequences.

Show MeSH