Identification of an intragenic ribosome binding site that affects expression of the uncB gene of the Escherichia coli proton-translocating ATPase (unc) operon.
The uncB gene codes for the a subunit of the Fo proton channel sector of the Escherichia coli F1 Fo ATPase. Control of expression of uncB appears to be exerted at some step after translational initiation. Sequence analysis by the perceptron matrices (G. D. Stormo, T. D. Schneider, L. Gold, and A. Ehrenfeucht, Nucleic Acids Res. 10:2997-3011, 1982) identified a potential ribosome binding site within the uncB reading frame preceding a five-codon reading frame which is shifted one base relative to the uncB reading frame. Elimination of this binding site by mutagenesis resulted in a four- to fivefold increase in expression of an uncB'-'lacZ fusion gene containing most of uncB. Primer extension inhibition (toeprint) analysis to measure ribosome binding demonstrated that ribosomes could form an initiation complex at this alternative start site. Two fusions of lacZ to the alternative reading frame demonstrated that this site is recognized by ribosomes in vivo. The results suggest that expression of uncB is reduced by translational frameshifting and/or a translational false start at this site within the uncB reading frame.
Matten
S R
SR
Department of Biochemistry and Molecular Biology, Wayne State University School of Medicine, Detroit, Michigan 48201, USA.
Schneider
T D
TD
Ringquist
S
S
Brusilow
W S
WS
D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics; D025243:Bacterial Proton-Translocating ATPases; D001483:Base Sequence; D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics*; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D007438:Introns; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016571:Neural Networks, Computer; D009690:Nucleic Acid Conformation; D009876:Operon; D010957:Plasmids; D014176:Protein Biosynthesis; D012323:RNA Processing, Post-Transcriptional; D012333:RNA, Messenger / Q000737:chemistry* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012270:Ribosomes / Q000378:metabolism*; D001616:beta-Galactosidase / Q000096:biosynthesis
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D001616:beta-Galactosidase; D025243:Bacterial Proton-Translocating ATPases; C074428:atpB protein, E coli