# Validation of Omega Subunit of RNA Polymerase as a Functional Entity
## 7 Role of \(\omega\)-Subunit in Stringent Response  
The _rpoZ_ gene encoding for \(\omega\)-subunit in _E. coli_, is located in the same operon as _spoT_. The product of _spoT_ gene is responsible for the synthesis and degradation of bacterial alarmone molecules known as ppGpp [43]. Guanosine tetrapphosphate (ppGpp) is a bacterial alarmone that enables bacterial cells to swiftly reprogram their gene expression to adapt various stress conditions. ppGpp and its precursor guanosine pentaphosphate (pppGpp), collectively known as (p)ppGpp or magic spot, were discovered more than 50 years ago in _Escherichia coli_ during amino acid starvation experiments, and they are  
Figure 3: \(\omega\), \(\beta\)’ interaction and its schema. (**A**) Interaction between \(\omega\) and \(\beta\)’ subunit in bacterial RNAP. As shown in the figure, \(\omega\) (cyan) interacts with \(\beta\)’ (green) via salt bridges and hydrogen bonding (black dotted line) between the side chains. The amino acids of \(\omega\) interacting with the \(\beta\)’ subunit play an important role in maintaining the structural integrity of the protein. Change in the structural content of \(\omega\) would interfere with the native interactions. (**B**) \(\omega\) protein alone is an unstructured protein (green) that undergoes structured transition to ordered structure (blue) upon reconstitution to functional RNAP. Dominant lethal mutant of “\(\omega\)” (red), point or silent in nature are structured, to begin with which affects the plasticity of the RNAP active site upon reconstitution. known as effectors for stringent response [44]. Although the effect of ppGpp is broadly manifested in bacterial physiology and during regulation of replication and translation, it has a profound effect on transcription rates, like the repression of stable RNA synthesis and the induction of stress response factors and genes that are required for amino acid biosynthesis and transport. Genomic profiling showed that the stringent response alters the expression of hundreds of genes [45; 46; 47; 48; 49]. However, the most interesting observation that came from the crystal structure of RNAP with ppGpp, was that the primary site for ppGpp binding is located at \(\beta^{\prime}\)-\(\omega\) interface in _E. coli_[35]. When the activity of the enzyme was tested for ppGpp with and without \(\omega\)-subunit in an in-vitro transcription reaction, it was observed that \(\omega\)-plays a role in regulating the sensitivity of the enzyme to ppGpp [50; 51]. However, it was further debated when the \(\Delta rpoZ\) strain of _E. coli_, showed a normal stationary phase response [52]. Although these observations are contradictory to each other, it can be assumed that there must be a redundancy in the role of \(\omega\) in the stringent response. The aforementioned statement can be supported with the observation that a second binding site for ppGpp with RNAP is available in the presence of DksA transcription factor [53; 54]. This second binding site is more critical to stringent response than the first one [55]. The primary function of \(\omega\) has been recognized as to provide the scaffold for \(\beta^{\prime}\)-subunit to fold correctly and to latch it on the \(\alpha 2\beta\) subassembly. An earlier study by our group showed that the \(\omega\)-less RNAP has lower affinity to DNA in comparison to the affinity with native RNAP [25]. It had been reported that in \(\omega\)-less strain of _E. coli_, the content of the stationary phase sigma factor \(\sigma\)S, increased in comparison to that of \(\sigma 70\)[56]. In accordance with the decrease in the amount of RNAP-\(\sigma 70\) holoenzyme, the \(\Delta rpoZ\) strain of _E. coli_ showed reduced growth rate at standard growth conditions. However, the slowing down of growth could also be due to the polarity effect on the \(spoT\) gene located downstream to the \(rpoZ\) in the same operon.