Polarity Effects in the Lactose Operon of Escherichia coli

Abstract

An intergenic RNA segment between lacY and lacA of the lactose operon in Escherichia coli is cleaved by RNase P, an endoribonuclease. The cleavage of the intergenic RNA was ten times less efficient than cleavage of a tRNA precursor in vitro. Fragments of the RNase P cleavage product are detectable in vivo in the wild-type strain but not in a mutant strain at the restrictive temperature. The cleavage product that contains lacA in the wild-type strain was quickly degraded. When this intergenic segment was cloned upstream of a reporter gene, the expression of the reporter gene was also inhibited substantially in wild-type E. coli, but not in a temperature sensitive mutant strain in RNase P at the restrictive temperature. These results support data regarding the natural polarity between lacZ versus lacA, the downstream gene.

Introduction

The lactose (lac) operon in Escherichia coli has been regarded for four decades as a paradigm for the understanding of the molecular biology of bacterial gene expression and its regulation. The basic scheme of the lac operon is illustrated in Figure 1A. The cluster of three structural genes, lacZ (β-galactosidase), lacY (lactose permease), and lacA (transacetylase), is flanked by a promoter and a terminator to signal the beginning or ending of transcription.¹ There are still some remaining uncertainties about regulation in the lac operon. In either high or low β-galactosidase producing strains, approximately three to five times more β-galactosidase monomers are synthesized than transacetylase monomers.² These data reveal that the overall rate of translation is at least ten time higher from lacZ than from lacA when the operon is induced. This natural polarity is absent in other well-known operons such as trp and his, in which all peptides from structural genes are synthesized in about equimolar amounts.2., 3.

Careful examination of recent high-density DNA microarray experiments (41 sets of data) from the E. coli Functional Genomics Project† also reveals that the mRNA levels of lacZ are three to four times higher than those of lacA, regardless of the various conditions used for growth of E. coli or its mutant derivatives thereof. The microarrays were probed with a fixed number of oligonucleotides to every total gene sequence. It has been found that the lacA mRNA “decays” faster that lacZ mRNA,⁴ which could be a possible means of accounting for the natural polarity in the lac operon. Other general speculative hypotheses for differing molar concentrations of peptides encoded in the same operon have been suggested, but none of them could provide a satisfactory explanation given that there were no additional experimental data about this operon available.

In E. coli, RNase P cleaves the intergenic transcript of the tna operon, which consists of the tnaL, tnaA and tnaB genes.⁵ The tnaB mRNA is always significantly lower than that of tnaA mRNA. Other operons are also cleaved in certain intergenic regions. The action of RNase P and subsequent RNase E degradation of the fragments downstream from RNase P cleavage site of the polycistronic mRNA are thought to be responsible for the natural polarity of tnaA and tnaB.5., 6. In this work, the intergenic transcript lacZY (the fragment between lacZ and lacY) and lacYA (the fragment between lacY and lacA) were exposed to RNase P to determine whether RNA segments are substrates for enzyme. We found that the lacYA RNA segment could be cleaved by E. coli RNase P holoenzyme, as well as by the RNA subunit, itself, in vitro. This cleavage does not take place under restrictive conditions in a strain thermosensitive for RNase P activity. The intergenic RNA segment, lacYA, may be responsible for the relatively lower expression of lacA by serving as a destabilizing element of downstream lacA mRNA.