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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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Electrophoretic mobility shift analysis of the interaction between the variants of the SacT CAT with the different RAT structures. The different CAT domains [(A) GlcT (B) SacT (C) SacT P26R, Q31R (D) SacT P26C, Q31R (E) SacT P26S, Q31] were tested against the RAT RNAs (1: ptsG; 2: licTopt; 3: sacB; 4: bglPR 5: sacPR). A 100 pmol of the RAT–RNAs were used. In lanes labelled with ‘+’ 250 pmol of the RNA-binding domain was added to the RNA as indicated prior to electrophoresis.
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Figure 3: Electrophoretic mobility shift analysis of the interaction between the variants of the SacT CAT with the different RAT structures. The different CAT domains [(A) GlcT (B) SacT (C) SacT P26R, Q31R (D) SacT P26C, Q31R (E) SacT P26S, Q31] were tested against the RAT RNAs (1: ptsG; 2: licTopt; 3: sacB; 4: bglPR 5: sacPR). A 100 pmol of the RAT–RNAs were used. In lanes labelled with ‘+’ 250 pmol of the RNA-binding domain was added to the RNA as indicated prior to electrophoresis.

Mentions: The RNA-binding domain of GlcT did only recognize its cognate RAT, ptsG (Figure 3A). This is in excellent agreement with our previous observations. As shown in Figure 3B, the CAT of SacT was able to retard the migration of its cognate sacPA RAT. In addition, weak retardation of the sacB RAT was observed. This is in good agreement with the observation that the sacB RAT structure is a poor target for the naturally occurring anti-terminator proteins (see above). The three selected CAT variants (originally encoded on pGP451, pGP452 and pGP453; see Table 3) retarded all the tested RAT RNAs (Figure 3C–E). This confirms the loss of RNA recognition specificity already observed in the in vivo anti-termination assay.Figure 3.


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)

Electrophoretic mobility shift analysis of the interaction between the variants of the SacT CAT with the different RAT structures. The different CAT domains [(A) GlcT (B) SacT (C) SacT P26R, Q31R (D) SacT P26C, Q31R (E) SacT P26S, Q31] were tested against the RAT RNAs (1: ptsG; 2: licTopt; 3: sacB; 4: bglPR 5: sacPR). A 100 pmol of the RAT–RNAs were used. In lanes labelled with ‘+’ 250 pmol of the RNA-binding domain was added to the RNA as indicated prior to electrophoresis.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3105407&req=5

Figure 3: Electrophoretic mobility shift analysis of the interaction between the variants of the SacT CAT with the different RAT structures. The different CAT domains [(A) GlcT (B) SacT (C) SacT P26R, Q31R (D) SacT P26C, Q31R (E) SacT P26S, Q31] were tested against the RAT RNAs (1: ptsG; 2: licTopt; 3: sacB; 4: bglPR 5: sacPR). A 100 pmol of the RAT–RNAs were used. In lanes labelled with ‘+’ 250 pmol of the RNA-binding domain was added to the RNA as indicated prior to electrophoresis.
Mentions: The RNA-binding domain of GlcT did only recognize its cognate RAT, ptsG (Figure 3A). This is in excellent agreement with our previous observations. As shown in Figure 3B, the CAT of SacT was able to retard the migration of its cognate sacPA RAT. In addition, weak retardation of the sacB RAT was observed. This is in good agreement with the observation that the sacB RAT structure is a poor target for the naturally occurring anti-terminator proteins (see above). The three selected CAT variants (originally encoded on pGP451, pGP452 and pGP453; see Table 3) retarded all the tested RAT RNAs (Figure 3C–E). This confirms the loss of RNA recognition specificity already observed in the in vivo anti-termination assay.Figure 3.

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