Mapping of sequences required for the translation of the β subunit ofEscherichia coliRNA polymerase

Abstract

Previous experiments using expression plasmids which overproduce the β and β′ subunits of Escherichia coli RNA polymerase suggested that regions considerably upstream of the start of the rpoB gene, which encodes the β subunit, are required for its efficient synthesis. To further delineate the required regions, a collection of genetic constructs that contained varying amounts of the region either upstream or downstream of the translational start of rpoB was assembled. Measurements of β and β′ synthesis and rpoB mRNA production from a series of rpoBC expression plasmids indicated that sequences extending more than 43 bp but less than 79 bp upstream of rpoB are required for the efficient translation of rpoB mRNA. This result was confirmed by β-galactosidase measurements from a series of rpoB-lacZ fusions that have the same set of end points upstream of rpoB as the expression plasmids. A second set of gene fusions containing differing amounts of the sequence distal to the start of rpoB fused in frame to lacZ revealed that more than 29 bp but less than 70 bp of rpoB was required for efficient translation.Key words: RNA polymerase, E. coli, translational regulation.