RNA recognition by transcriptional antiterminators of the BglG/SacY family: functional and structural comparison of the CAT domain from SacY and LicT.
Transcriptional antiterminators of the BglG/SacY family are regulatory proteins that mediate the induction of sugar metabolizing operons in Gram-positive and Gram-negative bacteria. Upon activation, these proteins bind to specific targets in nascent mRNAs, thereby preventing abortive dissociation of the RNA polymerase from the DNA template. We have previously characterized the RNA-binding domain of SacY from Bacillus subtilis and determined its three-dimensional structure by both NMR and crystallography. In the present study, we have characterized the paralogous domain from LicT and we present the first structural comparison between two BglG/SacY family members. Similar to SacY, the RNA-binding activity of LicT is contained within the 56 N-terminal amino acid residue fragment corresponding to the so-called co-antiterminator (CAT) domain. Surface plasmon resonance affinity measurements show that, compared to SacY-CAT, LicT-CAT binds more tightly and more specifically to its cognate RNA target, with a KD value of about 10(-8) M. The crystal structure of LicT-CAT has been determined at 1.8 A resolution and compared to that of SacY-CAT. Both molecules fold as symmetrical dimers, each monomer comprising a four-stranded antiparallel beta-sheet that stacks against the beta-sheet of the other monomer in a very conserved manner. Comparison of the proposed RNA-binding surfaces shows that many of the conserved atoms concentrate in a central region across one face of the CAT dimer, whereas variable elements are mostly found at the edges. Interestingly, the electrostatic potential maps calculated for the two molecules are quite different, except for the core of the RNA-binding site, which appears essentially neutral in both structures.
Declerck
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Architecture et Fonction des Macromolécules Biologiques, CNRS-UPR9039, 31 Chemin Joseph Aiguier, Marseille Cedex 20, F-13402, France. nathalie@esil.univ-mrs.fr
Vincent
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Hoh
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Aymerich
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van Tilbeurgh
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H
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D018360:Crystallography, X-Ray / Q000379:methods; D019281:Dimerization; D008958:Models, Molecular; D008969:Molecular Sequence Data; D012313:RNA / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D020349:Surface Plasmon Resonance; D014157:Transcription Factors / Q000737:chemistry* / Q000378:metabolism; D014158:Transcription, Genetic
D001426:Bacterial Proteins; C098955:LicT protein, Bacteria; D016601:RNA-Binding Proteins; C076894:SacY protein, Bacillus subtilis; D014157:Transcription Factors; C064872:antiterminator proteins, Bacteria; D012313:RNA