# Validation of Omega Subunit of RNA Polymerase as a Functional Entity
## 4 The Role of \(\omega\) Subunit  
For a long period, probably until the late 1990s, the constitution of the core bacterial RNAP was considered as \(\alpha 2\beta\beta^{\prime}\). Protocols were designed to remove \(\omega\) subunit from RNAP purification and improve the purity of the enzyme [14]. Understanding the roles of individual subunits of RNAP is a prerequisite to follow the complex mechanism of transcription. The functional characterization using the biochemical and genetic tool for RNAP has assigned specific roles to the different subunits, except for the smallest subunit, \(\omega\), whose role in transcription remained elusive [15; 16]. Initial experiments showed that the \(\omega\)-less RNA polymerase could be reconstituted in vitro from individual subunits [17]. Moreover, concerning the wild-type strain, the \(\omega\) knockout strain of _Escherichia coli_ showed retarded growth but appeared morphologically the same, implying that \(\omega\) may not be an essential subunit of RNAP [2; 18]. What followed was a period of considerable lull as far as the \(\omega\) subunit was concerned, although the research on RNAP kept accelerating. This period was interspersed with cases where afew interesting observations about this small protein kept cropping up, such as identifying two \(\omega\) subunits from _Bacillus subtilis_. \(\omega 1\), which is now called epsilon, and \(\omega 2\), is a homolog of the \(\omega\) protein from _E. coli_ and other bacteria [19; 20]. But the debate on whether \(\omega\) was a subunit of the enzyme or a protein contaminant remained alive until recent times. Meanwhile, the possibility that \(\omega\) could still be a subunit of RNAP received a fillip when the work described by [PERSON] and [PERSON] [21] showed that the majority of \(\omega\) protein in _E. coli_ cell lysate remained bound to RNAP and a peptide of similar size cross-reacting with anti-\(\omega\) antibodies was present in several enteric bacteria. Subsequently, two articles were published, which questioned the redundancy of \(\omega\) being merely an abundant contaminant. First was the convincing identification of \(\beta^{\prime}\) subunit as the binding site of \(\omega\) using cross-linking studies [21]. The second set of elegant experiments utilized the natural phenomenon of transcription activation by protein-protein interaction when one of the interacting proteins was tethered to DNA, and the other is a subunit of RNAP. It was shown that a chimera of \(\omega\) and the \(\lambda\)CI repressor if expressed within a bacterium harboring a \(\lambda\) operator appropriately placed upstream to the promoter, could drive transcription from the promoter [22]. Based on these two results, the indecisiveness of whether the protein was a subunit of RNAP marked a turning point in the research on \(\omega\) subunit. The attempts to functionally characterize \(\omega\) subunit as a part of RNAP in bacteria finally bore fruit when [PERSON] and others [22; 23; 24; 25] provided the first indication of the possible function of \(\omega\) in RNAP in _E. coli_. They reported that when RNAP was purified from _rpoZ_-null strain of _E. coli_, the enzyme was found to be less than 50% activity compared to that of the wild-type enzyme. Another observation they made was that when the \(\omega\)-less enzyme was subjected to denaturation using 6M urea, the recovery of the enzyme increased upon the addition of \(\omega\) during reconstitution. Moreover, they also observed that in the absence of \(\omega\), RNAP co-purified with another large protein. It was later identified as GroEL. This led to interesting possibilities in the light of another almost simultaneous observation that \(\beta^{\prime}\) was the only subunit of _E. coli_ RNAP that was not folded by GroEL in vivo [26]. Association of GroEL with RNAP [27; 28] had been previously reported, but the report of [PERSON] et al. suggested a functional role of GroEL, in the folding of all the subunits of RNAP except \(\beta^{\prime}\), and in their assembly into the final enzyme [25].