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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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Structure of the LicT-CAT/licS-RAT complex showing amino acid residues targeted for mutagenesis. The dimeric structure of the LicT N-terminal domain (residues 1–56) determined by NMR (17, PDB entry code 1L1C) is shown in cartoon and surface representation, with one monomer coloured in pink and the other in green. The amino acid side-chains of the residues targeted for site-directed mutagenesis in this study as well as a key residue of CAT–RAT interaction (Phe31) are labelled and shown in sticks. The licS RAT RNA is shown in wire frame with the phosphate backbone cartooned in pink for internal loop 1, green for internal loop 2 and orange elsewhere. In the LicT CAT-RAT structure, A26 in loop 1 and U8 in loop 2 are bulged out from the RNA helix core and are recognized by symmetry-related elements of the LicT-CAT dimer interface.
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Figure 5: Structure of the LicT-CAT/licS-RAT complex showing amino acid residues targeted for mutagenesis. The dimeric structure of the LicT N-terminal domain (residues 1–56) determined by NMR (17, PDB entry code 1L1C) is shown in cartoon and surface representation, with one monomer coloured in pink and the other in green. The amino acid side-chains of the residues targeted for site-directed mutagenesis in this study as well as a key residue of CAT–RAT interaction (Phe31) are labelled and shown in sticks. The licS RAT RNA is shown in wire frame with the phosphate backbone cartooned in pink for internal loop 1, green for internal loop 2 and orange elsewhere. In the LicT CAT-RAT structure, A26 in loop 1 and U8 in loop 2 are bulged out from the RNA helix core and are recognized by symmetry-related elements of the LicT-CAT dimer interface.

Mentions: Examination of the homologous LicT CAT–RAT complex structure (Figure 5) shows that the corresponding residues in LicT (Arg-27 and Gln-32) are located on the outer edges of the RNA-binding surface of the protein dimer. The side-chains of these polar residues form like two grips, each contacting one strand of the RNA stem flanking the two internal loops (1 and 2) characterizing the RAT hairpin (see also Figure 1A). Although different in sequence, these loops present analogous 3D structures and can therefore be recognized in a similar way by symmetry-related elements of the LicT CAT dimer (15). In particular, A26 in loop 1 and U8 in loop 2, which are major specificity determinants of the RAT RNAs, are both expelled from the core of the RNA helix and their base is similarly accommodated within a cavity formed on each side of the dimer interface. The side-chain of Arg-27 contributes to the formation of these cavities and adopts a slightly different conformation in the two CAT monomers in order to optimize contacts with the bulged-out pyrimidine or purine, as well as with the sugar phosphate backbone. On the other side of the RNA minor groove Gln-32 is interacting with the phosphate group of U4 and C23, but also with the aromatic side-chain of the strictly conserved phenylalanine at position 31, which is crucial for the formation and stabilization of the sheared base pairs in both loops 1 and 2.Figure 5.


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)

Structure of the LicT-CAT/licS-RAT complex showing amino acid residues targeted for mutagenesis. The dimeric structure of the LicT N-terminal domain (residues 1–56) determined by NMR (17, PDB entry code 1L1C) is shown in cartoon and surface representation, with one monomer coloured in pink and the other in green. The amino acid side-chains of the residues targeted for site-directed mutagenesis in this study as well as a key residue of CAT–RAT interaction (Phe31) are labelled and shown in sticks. The licS RAT RNA is shown in wire frame with the phosphate backbone cartooned in pink for internal loop 1, green for internal loop 2 and orange elsewhere. In the LicT CAT-RAT structure, A26 in loop 1 and U8 in loop 2 are bulged out from the RNA helix core and are recognized by symmetry-related elements of the LicT-CAT dimer interface.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 5: Structure of the LicT-CAT/licS-RAT complex showing amino acid residues targeted for mutagenesis. The dimeric structure of the LicT N-terminal domain (residues 1–56) determined by NMR (17, PDB entry code 1L1C) is shown in cartoon and surface representation, with one monomer coloured in pink and the other in green. The amino acid side-chains of the residues targeted for site-directed mutagenesis in this study as well as a key residue of CAT–RAT interaction (Phe31) are labelled and shown in sticks. The licS RAT RNA is shown in wire frame with the phosphate backbone cartooned in pink for internal loop 1, green for internal loop 2 and orange elsewhere. In the LicT CAT-RAT structure, A26 in loop 1 and U8 in loop 2 are bulged out from the RNA helix core and are recognized by symmetry-related elements of the LicT-CAT dimer interface.
Mentions: Examination of the homologous LicT CAT–RAT complex structure (Figure 5) shows that the corresponding residues in LicT (Arg-27 and Gln-32) are located on the outer edges of the RNA-binding surface of the protein dimer. The side-chains of these polar residues form like two grips, each contacting one strand of the RNA stem flanking the two internal loops (1 and 2) characterizing the RAT hairpin (see also Figure 1A). Although different in sequence, these loops present analogous 3D structures and can therefore be recognized in a similar way by symmetry-related elements of the LicT CAT dimer (15). In particular, A26 in loop 1 and U8 in loop 2, which are major specificity determinants of the RAT RNAs, are both expelled from the core of the RNA helix and their base is similarly accommodated within a cavity formed on each side of the dimer interface. The side-chain of Arg-27 contributes to the formation of these cavities and adopts a slightly different conformation in the two CAT monomers in order to optimize contacts with the bulged-out pyrimidine or purine, as well as with the sugar phosphate backbone. On the other side of the RNA minor groove Gln-32 is interacting with the phosphate group of U4 and C23, but also with the aromatic side-chain of the strictly conserved phenylalanine at position 31, which is crucial for the formation and stabilization of the sheared base pairs in both loops 1 and 2.Figure 5.

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