From genetic to structural characterization of a new class of RNA-binding domain within the SacY/BglG family of antiterminator proteins.
SacY is the prototype of a family of regulatory proteins able to prevent transcription termination. It interacts with a 29 nucleotide RNA sequence able to fold into a stem-loop structure and partially overlapping with a terminator sequence located in the 5' leader mRNA region of the gene it controls. We show here that the N-terminal fragment of SacY, SacY(1-55), and the corresponding fragments of other members of the family have antiterminator activities with efficiency and specificity identical to those of the full-length proteins. In vitro, this activity correlates with the specific affinity of SacY(1-55) for its RNA target. UV melting experiments demonstrate that SacY(1-55) binding stabilizes the RNA target structure. The NMR solution structure of SacY(1-55) is very similar to that obtained in the crystal (van Tilbeurgh et al., 1997): the peptide is folded as a symmetrical dimer without any structural homology with other RNA-binding domains yet characterized. According to a preliminary NMR analysis of the SacY(1-55)-RNA complex, the protein dimer is not disrupted upon RNA binding and several residues implicated in RNA recognition are located at the edge of the dimer interface. This suggests a new mode of protein-RNA interaction.
Manival
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CBS, Faculté de Pharmacie, CNRS-UMR9955, INSERM-U414, Université Montpellier, France.
Yang
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Strub
M P
MP
Kochoyan
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Steinmetz
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M
Aymerich
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S
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002850:Chromatography, Gel; D019281:Dimerization; D017930:Genes, Reporter; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D011487:Protein Conformation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D012333:RNA, Messenger / Q000737:chemistry / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D014157:Transcription Factors
D001426:Bacterial Proteins; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; C076894:SacY protein, Bacillus subtilis; D014157:Transcription Factors; C064872:antiterminator proteins, Bacteria