# Validation of Omega Subunit of RNA Polymerase as a Functional Entity
## 6 Structure-Function Relationship in \(\omega\)-Subunit  
At this stage it was important to evaluate the physical parameters that govern the interaction between mutant or wild-type \(\omega\) and subassembly \(\alpha 2\beta\beta\)' and subsequent recognition of \(\alpha 2\beta\beta\)'\(\omega\) with \(\sigma\)-subunit. Studies from our laboratory [33; 34; 38] have shown that dominant lethal mutant of \(\omega\)-subunit in _E. coli_ shows tighter binding with the RNAP (\(\alpha 2\beta\beta\)'), compared to that of wild-type \(\omega\). Thermodynamics of this recognition suggests that the interaction between structured \(\omega\) and \(\alpha 2\beta\beta\)' is driven by high negative enthalpy and a small but unfavorable negative entropy term. The key thermodynamic driving force for the binding reaction is generally a favorable enthalpy contribution. Analysis of crystal structure of RNA polymerase reveals the following details [35]. The interface area of \(\beta\)' and \(\omega\) is 1424.0 A2. There is a significant interaction between both the subunits and any change in \(\omega\) structure can change the parameters of interaction between the two subunits. Interaction between \(\beta\)' and \(\omega\) is guided by virtue of electrostatic interaction between the side chains and hydrogen bonding between the backbone residues (Figure 3A). The structured \(\omega\)-subunit binds to \(\alpha 2\beta\beta\)' subassembly, which is evident from in vitro reconstitution and surface plasmon resonance (SPR) or isothermal calorimetry (ITC) studies, but the reconstituted enzyme is defective during transcription initiation [33; 38]. However, if pre-initiated RNA is supplied to the reaction mixture, elongation of RNA continues unhindered in the presence of the mutated RNAP [33]. This again hints at the importance of the disordered region in \(\omega\). As recognized through X-ray structure of RNAP that \(\beta\)'-F-(bridge)-helix and \(\beta\)'-trigger loop encompasses the active center of RNA polymerase and they work in a coordinated fashion to permit the correct placement of NTPs at the active site resulting in their incorporation into the elongating transcripts [39; 40; 41; 42]. Thus, due to the binding of the structured \(\omega\), the mobility of the flexible segments at the active site is affected, which manifests into the abrogation of initiation of transcription. The structured \(\omega\)- affects the plasticity of the active site, which makes the RNAP initiation defective (Figure 3B). The function of RNAP depends on the movement of many inter and intra-segmental domains relative to each other, and the movement of all domains, including different subunits provides the skeleton of the complex. The major contributors to this structural flexibility of RNAP are the \(\beta\) and \(\beta\)'-subunit as they are the catalytic site of the enzyme [39]. Structural alterations in the catalytic subunit would hinder the molecular motion and would affect the functioning of RNAP. It appears that \(\omega\)-subunit, whose essential function is to provide a scaffold for one of the catalytic subunits \(\beta\)' plays a role that affects the movement of the domains at the RNAP active site. This was validated by mapping a suppressor mutant for structured \(\omega\), in \(\beta\)' subunit [33]. This \(\beta\)' suppressor mutant bears the Y457S substitution located immediately adjacent to the conserved motif NADFDGD, the active site residues on \(\beta\)'-subunit [33; 42]. This motif co-ordinate the catalytic Mg+2 ions before the transcription process starts. In an unpublished study from our group, the same suppressor mutant of \(\beta\)' subunit was used to rescue the effect of silent mutants of \(\omega\). It was observed that the same suppressor mutant of \(\beta\)'-subunit, which was able to compensate for a point mutant of \(\omega\) (N60D), was able to rescue the phenotype due to structured silent mutants of \(\omega\) as well.