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Function in vivo of separate segments of the β subunit of Escherichia coli RNA polymerase
Author(s) -
Trigwell Susan,
Glass Robert E.
Publication year - 1998
Publication title -
genes to cells
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.912
H-Index - 115
eISSN - 1365-2443
pISSN - 1356-9597
DOI - 10.1046/j.1365-2443.1998.00220.x
Subject(s) - biology , rna polymerase , specificity factor , polymerase , transcription (linguistics) , rna polymerase ii , protein subunit , microbiology and biotechnology , gene , structural gene , transcription factor ii d , rna polymerase i , rna , gene expression , genetics , escherichia coli , promoter , linguistics , philosophy
Background: Transcription of genetic material is catalysed by the enzyme DNA‐dependent, RNA polymerase. 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 & 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.