Physical mapping of a collection of MaeI-generating amber mutations in the β gene of Escherichia coli RNA polymerase and the functional effect of internal deletions constructed through their manipulation

Abstract

We report the fine mapping of 55 of our 95 amber mutations in the β gene of Escherichia coli RNA polymerase by virtue of the unique MaeI restriction sites created by this subset of nonsense mutations (i.e. CTAG, where the amber codon is underlined). [The full data are reported in Supplementary Publication SUP 50181 (12 pages), which has been deposited at the British Library Document Supply Centre, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1997) 321, 8–10.] The CTAG mutations, which have been positioned to within approx. 9–60 bp, are distributed almost along the entire length of the rpoB gene, the one exception being the interval 400–499. The lack of amber fragments for mutations within the 5′ approx. 265 codons suggests lability of the extreme N-terminal region; further potential destabilizing ‘signals’ may be present in the non-conserved ‘spacer’ regions. The locations of four of the eleven rpoB amber mutations that are strongly polar on expression of the downstream rpoC gene have been determined through a combination of MaeI mapping, PCR amplification and DNA sequencing. Surprisingly, one such mutant carries two tandem CTAG sites but is viable with three of the nonsense suppressors tested. These polar amber sites define three different amino acids (Gln-31, Gln-83 and Trp-183) that fall within three sequence-conservation blocks in the N-terminal region. Six of the MaeI/Am (where MaeI/Am is an amber mutation generating a MaeI restriction site) rpoB alleles (Gln-83, Gln-276, Gln-327, Gln-618, Gln-649 and Trp-183) have been used to generate small in-frame deletions (31–100 codons) within conserved and non-conserved regions of the β gene, and the properties of these deletion variants were assessed in vivo. The smallest deletion reported in this study removes 31 amino acids from the middle of a region common to the eubacterial/chloroplast subgroup of β homologues, and our results strongly suggest β^{ΔQ618–Q649} is assembled into a holoenzyme form capable of transcriptional initiation in vivo.