Subunits of RNA polymerase in function and structure. II. Reconstitution of Escherichia coli RNA polymerase from isolated subunits

Ishihama, A.; Ito, K.

Journal of Molecular Biology 72(1): 111-123


ISSN/ISBN: 0022-2836
PMID: 4567397
DOI: 10.1016/0022-2836(72)90073-3
Accession: 044465235

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The DNA-dependent Rna polymerase of Escherichia coli was found to be reactivated after complete dissociation, into subunits by treatment with 6 m-urea. The presence of glycerol appears to be essential throughout the procedure for this re-activation. Recovery of enzyme activity also depends on the ionic strength during both dissociation and re-association. The best recovery was obtained at 0.2 m-KC1 during re-association, whereas in the absence of salt the recovery was less than 10%. However, when re-activation was carried out in the presence of DNA, enzyme activity could be mostly recovered in the absence of salt though higher ionic strength was rather inhibitory. Thus, it was suggested that reconstitution of the polymerase from dissociated subunits might follow different steps depending upon whether it is Dna dependent or independent. In order to determine the molecular events in subunit assembly, individual subunits have been isolated by means of successive column chromatography on Sepharose 6B and DEAE-Sephadex A50. Possible formation of a binary complex was then examined for three different combinations: α- β, α- β- , and β- β- . Under the conditions used, a significant interaction was observed only between the α and β subunits, and the complex formed was found to have the structure α2β. Addition of the dialysed β- subunit converted this complex into the active enzyme. These results suggest that the assembly of subunits into Rna polymerase may be a sequential process with the formation of the α2β complex as an intermediate.