Small subunits of RNA polymerase: localization, levels and implications for core enzyme composition

Abstract

Bacterial RNA polymerases (RNAPs) contain several small auxiliary subunits known to co-purify with the coreα,βandβ′ subunits. Theωsubunit is conserved between Gram-positive and Gram-negative bacteria, while theδsubunit is conserved within, but restricted to, Gram-positive bacteria. Although various functions have been assigned to these subunits viain vitroassays, very little is known about theirin vivoroles. In this work we constructed a pair of vectors to investigate the subcellular localization of theδandωsubunits inBacillus subtiliswith respect to the core RNAP. We found these subunits to be closely associated with RNAP involved in transcribing both mRNA and rRNA operons. Quantification of these subunits revealedδto be present at equimolar levels with RNAP andωto be present at around half the level of core RNAP. For comparison, the localization and quantification of RNAPβ′ andωsubunits inEscherichia coliwas also investigated. Similar toB. subtilis,β′ andωclosely associated with the nucleoid and formed subnucleoid regions of high green fluorescent protein intensity, but, unlikeωinB. subtilis,ωlevels inE. coliwere close to parity with those ofβ′. These results indicate thatδis likely to be an integral RNAP subunit in Gram-positives, whereasωlevels differ substantially between Gram-positives and -negatives. Theωsubunit may be required for RNAP assembly and subsequently be turned over at different rates or it may play roles in Gram-negative bacteria that are performed by other factors in Gram-positives.