Temperature‐Sensitive Mutants of Escherichia coli with Defects in the Assembly of RNA Polymerase in vitro

RNA polymerase was purified from three temperature‐sensitive mutants of Escherichia coli , one (A2R7) mapping in rpoB , the second (R120) in rpoC , and the third (R60‐39), which has not been genetically mapped as yet, but which was found to produce a temperature‐sensitive β subunit. The enzymes were tested for their ability to reconstitute active enzyme from isolated subunits. RNA polymerase from A2R7 showed a reduced level of reconstitution compared to wild‐type enzyme at 30°C while no reconstitution was observed if the subunits were incubated at 42°C, the non‐permissive temperature for growth of the mutant bacteria. Wild‐type enzyme shows nearly full reconstitution at this temperature. Once assembled, the A2R7 holoenzyme complex is temperature resistant. The temperature‐sensitive lesion could be assigned to the β subunit, and it appears that the assembly is blocked primarily at a step involving the addition of the β′ subunit. The RNA polymerase of R120 fails to reconstitute to an active holoenzyme at any temperature. Mixed reconstitution experiments with wild‐type subunits showed that the subunit responsible for this lack of reconstitution is β′. The holoenzyme itself is temperature resistant. The RNA polymerase of R60‐39 assembles normally in vitro at 35°C but fails to assemble at 42°C. The temperature‐sensitive lesion of the RNA polymerase is due to its β′ subunit and, as in the case of A2R7, the assembly seems to be blocked at a step involving the addition of β′ to the α 2 β complex at 42°C. The holoenzyme itself shows only a slightly reduced rate of RNA synthesis at 42°C. We conclude that for all three mutants the temperature‐sensitive lesions in vivo are due to a failure of the newly synthesized polypeptide chains to form an active holoenzyme complex at the non‐permissive temperature.