Function in vivo of separate segments of the β subunit of Escherichia coli RNA polymerase

Abstract

Background: Transcription of genetic material is catalysed by the enzyme DNA-dependent, RNA polymeruse. The multimeric RNA polymerases consist of between 4 and 16 different subunits, of which the two largest, termed β and β', are conserved throughout nature. The β subunit has been implicated in all of the stages of transcription that are catalysed by the complete enzyme. Several lines of evidence have suggested that the function of the β subunit is not dependent upon the contiguity of the sequence blocks. In this report, a complementary immunological and genetic approach was adopted in order to investigate the individual regions of the β subunit of RNA polymerase. To this end, the β structural gene rpoB was separated into four near-equal, non-overlapping segments (as well as 'half' genes) on the basis of 'split' genes in nature, known functional organization and sequence conservation. These segments were used to prepare sequence-specific antibodies against the four individual regions, as well as being expressed in vivo from a tight, lac-controlled high-copy number vector. Results: Immunological probing of the holoenzyme in vitro suggested that the amino-terminal half of the β polypeptide is buried within the enzyme complex. Of the four segments expressed in vivo, the extreme C-terminal segment was trans-dominant lethal (of the effect of large N-terminal amber fragments on cellular growth; Nene and Glass 1982) and this isolated region was shown to bind the translational elongation factor EF-Tu in vivo. Conclusions: These in vivo and in vitro studies, in conjunction with recent in vitro work (Severinov et al. 1995), unambiguously demonstrate that individual regions of β may adopt structurally and functionally competent forms, and underline the possibility of in vivo investigation of separate regions of this massive polypeptide chain. A model is presented for the role of EF-Tu in stringent control.