Determinants of translational initiation efficiency in the atp operon of Escherichia coli

Abstract

Transcription and translation of the atp genes encoding the subunits b, delta, alpha, gamma and epsilon of the Escherichia coli H+-ATPase were studied. The nature and quantities of the respective transcripts initiated from different promoters were compared with overall expression rates thus yielding accurate information about relative translational efficiency and its coupling to mRNA levels. Part of the highly efficient subunit c gene translational initiation region (TIR) was used as a tool in manipulating the TIRs of the other genes. Rate control of atp cistron translation occurs at the initiation level and is determined locally by each gene's TIR. In this way, individual subunit synthesis rates are set to match the requirements for H+-ATPase assembly. There is no (or very restricted) translational coupling between the cistrons. Translational initiation rates of the normally weakly expressed atp genes could be increased by up to a factor of 27 by manipulating the sequences upstream of the start codons, despite biased codon usages. In the presence of an improved upstream sequence, the N-terminal sequence of the subunit gamma gene exerted a limiting effect. This could be relieved by altering the sequence of the first seven codons. The levels of subunit gamma mRNA were more sensitive to changes in translational efficiency than the concentrations of the other atp mRNAs. The relationships between initiation efficiency and primary and secondary structure in the natural and manipulated atp TIRs are discussed in detail.