Identification of an Intragenic Ribosome Binding Site That Affects Expression of the uncB Gene of the Escherichia coli Proton-Translocating ATPase ( unc ) Operon

Abstract

ABSTRACT The uncB gene codes for the a subunit of the F o proton channel sector of the Escherichia coli F 1 F o 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.