RNA recognition by transcriptional antiterminators of the BglG/SacY family: mapping of SacY RNA binding site.
Transcriptional antiterminators of the BglG/SacY family are bacterial regulatory proteins able to prevent the premature arrest of transcription through specific binding to a ribonucleic antiterminator (RAT) sequence. The RNA recognition module of these regulators is made of the 55-amino acid long N-terminal domain which can by itself promote efficient antitermination activity in vivo and RNA binding in vitro. The structure of this domain, which was called CAT for co-antiterminator, has first been determined for SacY from Bacillus subtilis and the putative surface contacting RNA has been defined by NMR footprinting. Here we have performed a genetic mapping of the SacY-CAT RNA binding site by substituting 24 amino acid residues including those previously identified by NMR, the highly conserved residues in the 55 homologous antiterminators recognised in the databases and all the positively charged residues. A total of 57 SacY-CAT variants have been constructed and tested in vivo for their antitermination efficiency. A few of these variants were then purified in order to analyse their RNA binding properties by surface plasmon resonance and to check their structural integrity by NMR. The present study validates and clarifies the RNA interacting surface previously mapped by NMR. The residues that are the most intolerant to substitutions, Asn8, His9, Asn10, Gly25, Gly27, and Phe30, are aligned across the CAT dimer interface and form the core of the RNA binding site. Three highly conserved residues stand outside the interaction surface but are essential for maintaining the CAT dimeric structure (Phe47) or may play an important functional role in the full length protein (Glu20 and Lys32). Interestingly, none of the twelve positively charged residues of SacY-CAT are crucial for the antitermination activity. By replacing three Lys residues and combining the Ala26-->Arg mutation that significantly enhanced the affinity for RNA, we engineered a SacY-CAT variant that should be suitable for NMR study of the complex.
Declerck
Nathalie
N
Génétique Moléculaire et Cellulaire, CNRS-URA1925, INRA-UMR216, INAP-G, F-78850 Thiverval-Grignon, France. nathalie@cbs.cnrs.fr
Minh
Nguyet Le
NL
Yang
Yinshang
Y
Bloch
Vanessa
V
Kochoyan
Michel
M
Aymerich
Stéphane
S
D000595:Amino Acid Sequence; D001120:Arginine / Q000235:genetics / Q000378:metabolism; D001412:Bacillus subtilis / Q000737:chemistry*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D017124:Conserved Sequence / Q000235:genetics; D019281:Dimerization; D008239:Lysine / Q000235:genetics / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D005810:Multigene Family; D016296:Mutagenesis / Q000235:genetics; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D012313:RNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D020349:Surface Plasmon Resonance; D013728:Terminator Regions, Genetic / Q000235:genetics*; D014157:Transcription Factors
D001426:Bacterial Proteins; D016601:RNA-Binding Proteins; C076894:SacY protein, Bacillus subtilis; D014157:Transcription Factors; C064872:antiterminator proteins, Bacteria; D012313:RNA; D001120:Arginine; D008239:Lysine