Subunit assembly in vivo of Escherichia coli RNA polymerase: role of the amino-terminal assembly domain of alpha subunit

Background: The RNA polymerase core enzyme of Escherichia coli is assembled in the sequence α→α₂→α₂β→α₂ ββ′. The amino-terminal domain down to residue 235 of the Escherichia coli RNA polymerase α subunit plays a key role in enzyme assembly. In vitro reconstitution studies from mutant α subunits have indicated the involvement of multiple sites for α dimerization, two regions (one near residue 45 and the other near residue 80) for β association, and two regions (one around residue 80 and the other between residues 180 and 200) for β′ association. The mechanism of RNA polymerase assembly in vivo, however, remains largely unknown. Results: RNA polymerase assembly in vivo was analysed for E. coli strains carrying expression plasmids for four amino-terminal deletion and 11 Ala-Ser (AS) dipeptide-insertion mutant α subunits. For detection of RNA polymerase complexes, a hexa-histidine (H₆) tag was added to all these mutant α at their carboxy-termini, and subunit complexes containing the H₆-tagged α were isolated by passing cell extracts through Ni²⁺-affinity columns. The assembly properties of most α mutants were consistent with those observed in in vitro reconstitution studies. Some mutants defective in β′ association in vitro such as those carrying mutations at residues 80 and 200 were, however, assembled in vivo, suggesting that a specific condition(s) or factor(s) supports RNA polymerase assembly in vivo. One possible candidate supporting the RNA polymerase assembly is the molecular chaperon(s), because DnaK (hsp70) was always associated with assembly-defective RNA polymerase mutants. Most assembly competent mutants complemented two temperature-sensitive mutant alleles of rpoA, but two assembly competent mutants, one (αΔN20) carrying a deletion at the extreme amino-terminal region and the other (αI-60) with AS insertion at residue 60, failed to complement these ts mutants. The failure suggests that these assembly competent but complementation-negative α mutants lack an as yet unidentified function(s). In the case of these two mutants, DnaK was associated, with apparently assembled RNA polymerase. Conclusion: The α–α, α–β and α–β′ contact sites on the RNA polymerase α subunit identified in in vitro reconstitution studies also participate in the subunit assembly in vivo. Some α mutants defective in assembly in vitro are, however, assembled in vivo. A factor(s) such as the molecular chaperon DnaK or a specific intracellular condition(s) may affect RNA polymerase assembly in vivo.