Abstract The C-terminal domain of the Escherichia coli RNA polymerase α subunit (αCTD) plays a key role in transcription initiation at many activator-dependent promoters and at UP element-dependent promoters. This domain is connected to the α N-terminal domain (αNTD) by an unstructured linker. To investigate the requirements of the α inter-domain linker to support growth of E. coli, we utilised a recently described technique for the substitution of the chromosomal rpoA gene, encoding α, by mutant rpoA alleles. We found that it was possible to replace wild-type rpoA by mutant alleles encoding α subunits containing inter-domain linkers that were longer by as many as 16 amino acids. However, using this method, it was not possible to transfer to the chromosome rpoA alleles encoding α subunits that contained an insertion of 32 amino acids or short deletions within the inter-domain linker. The effect of lengthening the α linker on activator-dependent and UP element-dependent transcription in the “haploid” rpoA system was shown to be qualitatively the same as observed previously in the diploid system. The ability of E. coli to tolerate insertions within the α inter-domain linker suggests that lengthening the α linker does not severely impair transcription of essential genes.