Identification of elements on GeneX-secA RNA of Escherichia coli required for SecA binding and secA auto-regulation.
The protein translocation ATPase of Escherichia coli, SecA protein, auto-regulates its translation by binding to its translation initiation region in geneX-secA mRNA. To analyze this regulation further the secondary structure of this portion of geneX-secA RNA was investigated utilizing structure-specific nucleases and chemical probing approaches. The results of this analysis were consistent with the existence of two adjacent helices, helix I and the lower portion of helix II, whose function in secA activation and repression, respectively, has been demonstrated. Binding of SecA protein to geneX-secA RNA or various mutant derivatives of this RNA was studied by measurement of affinity constants, RNA footprint analysis, and quantitation of auto-repression in vivo. This analysis showed that the SecA-binding site in geneX-secA RNA was remarkably large spanning a region of 96 nucleotides including a 3' portion of helix II, the secA translation initiation region and distal sequences. From the size of the SecA-binding site and the plasticity of its response to mutational alteration, it is suggested that SecA protein contains two distinct RNA-binding sites. Finally, it was shown that SecA binding was not sufficient to promote auto-regulation and that sequences both upstream (helix I) and within the binding site can contribute to auto-regulation without affecting SecA-binding affinity.
Salavati
R
R
Department of Molecular Biology and Biochemistry, Wesleyan University, Middletown, CT 06459, USA.
Oliver
D
D
D000251:Adenosine Triphosphatases / Q000235:genetics* / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D026901:Membrane Transport Proteins; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D014176:Protein Biosynthesis; D012329:RNA, Bacterial / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000069816:SEC Translocation Channels; D000081416:SecA Proteins; D017384:Sequence Deletion
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; D012329:RNA, Bacterial; D012333:RNA, Messenger; D000069816:SEC Translocation Channels; D000251:Adenosine Triphosphatases; D000081416:SecA Proteins