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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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Analysis of the SacT P26S mutant. The isolated SacT P26S variant was tested against all RAT structures in B. subtilis. The wild-type RNA-binding domains of SacT, LicT and GlcT were used as controls. The β-galactosidase activity is shown in percentage of the wild type activity. In the case of bglPR RAT SacT was set 100% because in this artificial system SacT has a higher activity towards this RAT structure. 1: SacT 2: LicT 3: GlcT 4: SacT P26S.
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Figure 2: Analysis of the SacT P26S mutant. The isolated SacT P26S variant was tested against all RAT structures in B. subtilis. The wild-type RNA-binding domains of SacT, LicT and GlcT were used as controls. The β-galactosidase activity is shown in percentage of the wild type activity. In the case of bglPR RAT SacT was set 100% because in this artificial system SacT has a higher activity towards this RAT structure. 1: SacT 2: LicT 3: GlcT 4: SacT P26S.

Mentions: Next, we wished to study whether the mutation in the CAT of SacT results in a complete switch of specificity, or in a relaxed RNA recognition. For this purpose, we used a set of reporter strains with lacZ fusions under the control of the different RATs. The results are shown in Figure 2. As expected, the licTopt RAT structure present in strain GP61 was recognized by LicT but not by SacT or GlcT. However, the isolated P26S variant of SacT was able to cause anti-termination at this structure. Similarly and in agreement with the data shown in Table 2, the sacPR RAT was specifically recognized by SacT. Interestingly, the SacT P26S variant CAT was able to anti-terminate at this structure; however, the activity was reduced to about one-third as compared to the wild-type CAT of SacT. The RAT structure of the sacB gene present in GP440 was a target of SacT. This RNA was also efficiently recognized by the SacT P26S CAT domain. The bglPR RAT is identical to the natural RNA structure of the bglPH operon. As shown previously, this RAT was recognized by both the SacT and the LicT CATs (20) whereas the CAT of GlcT was unable to cause anti-termination at this RNA structure. Interestingly, the SacT P26S CAT allows even higher anti-termination at this RAT than the wild-type RNA-binding domains. The last RAT structure in this study was that of ptsG present in B. subtilis GP109. This RAT was recognized by GlcT but neither by LicT nor by SacT. This is in good agreement with previous reports (16,18). The analysis of the activity of the SacT P26S CAT at this structure revealed that the mutant form of SacT is able to cause some anti-termination at this RAT (Table 2). This is the first time that a CAT domain different from that of GlcT was found to bind a ptsG RAT structure.Figure 2.


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)

Analysis of the SacT P26S mutant. The isolated SacT P26S variant was tested against all RAT structures in B. subtilis. The wild-type RNA-binding domains of SacT, LicT and GlcT were used as controls. The β-galactosidase activity is shown in percentage of the wild type activity. In the case of bglPR RAT SacT was set 100% because in this artificial system SacT has a higher activity towards this RAT structure. 1: SacT 2: LicT 3: GlcT 4: SacT P26S.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 2: Analysis of the SacT P26S mutant. The isolated SacT P26S variant was tested against all RAT structures in B. subtilis. The wild-type RNA-binding domains of SacT, LicT and GlcT were used as controls. The β-galactosidase activity is shown in percentage of the wild type activity. In the case of bglPR RAT SacT was set 100% because in this artificial system SacT has a higher activity towards this RAT structure. 1: SacT 2: LicT 3: GlcT 4: SacT P26S.
Mentions: Next, we wished to study whether the mutation in the CAT of SacT results in a complete switch of specificity, or in a relaxed RNA recognition. For this purpose, we used a set of reporter strains with lacZ fusions under the control of the different RATs. The results are shown in Figure 2. As expected, the licTopt RAT structure present in strain GP61 was recognized by LicT but not by SacT or GlcT. However, the isolated P26S variant of SacT was able to cause anti-termination at this structure. Similarly and in agreement with the data shown in Table 2, the sacPR RAT was specifically recognized by SacT. Interestingly, the SacT P26S variant CAT was able to anti-terminate at this structure; however, the activity was reduced to about one-third as compared to the wild-type CAT of SacT. The RAT structure of the sacB gene present in GP440 was a target of SacT. This RNA was also efficiently recognized by the SacT P26S CAT domain. The bglPR RAT is identical to the natural RNA structure of the bglPH operon. As shown previously, this RAT was recognized by both the SacT and the LicT CATs (20) whereas the CAT of GlcT was unable to cause anti-termination at this RNA structure. Interestingly, the SacT P26S CAT allows even higher anti-termination at this RAT than the wild-type RNA-binding domains. The last RAT structure in this study was that of ptsG present in B. subtilis GP109. This RAT was recognized by GlcT but neither by LicT nor by SacT. This is in good agreement with previous reports (16,18). The analysis of the activity of the SacT P26S CAT at this structure revealed that the mutant form of SacT is able to cause some anti-termination at this RAT (Table 2). This is the first time that a CAT domain different from that of GlcT was found to bind a ptsG RAT structure.Figure 2.

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