# Validation of Omega Subunit of RNA Polymerase as a Functional Entity
## 5 Functions of \(\omega\) Subunit in RNAP  
The X-ray crystal structure revealed many useful details. In many other prokaryotic organisms, it was observed that \(\omega\)-subunit plays the role of a molecular latch stabilizing the core enzyme structure and also increases the stability of the core complex [31]. Furthermore, through an extensive analysis of various genome sequences, it was revealed that the subunit homologues to \(\omega\) exist in archaeal RNAP (subunit RpoK) as well as in eukaryotic RNAPs I, II, and III (subunit RPB6) and their presence in archaea or eukaryotes is essential [31]. When we checked for sequence homology in _rpoZ_, we observed that in gram-positive and in gram-negative bacteria, homologous sequences are present as shown in Figure 1. Detailed sequence alignment revealed that there are three conserved regions, namely, CR1, CR2, and CR3, which are necessary for its function [30].  
Pseudomonas -MARYVEDCLINDVONFRELMALTKRADLAT6 GK- -P 37 Vibrio -MARYVUODAVEKIGNFRDLVLVARARARARQMC5 GK- -A 37 Pectobacterium -MARYVUODAVEKIGNFRDLVLVARARARQMC10 GGKD- -P 37 Escherichia -MARYVUODAVEKIGNFRDLVLVARARARQMC9 GK- -P 37 Salmonella -MARYVUODAVEKIGNFRDLVARARARQMC9 GK- -P 37 Streptromyces -MS55 ISAPEGIIINPPIDELLELAT5 YSKYLYAYARARQINMY5 QL  
rendered RNA polymerase defective during initiation of transcription. It should be mentioned here that the \(\omega\)-subunit exists as a well-folded structure in the crystal structure PDB (id: 4 JKR) of _E. coli_ RNA polymerase (Figure 2B) [35]. This is one characteristic of intrinsically disordered protein (IDP), where binding coupled folding for the protein takes place upon interaction with its partner/partners [36]. However, we are not sure at this stage whether a protein with only 91 amino acids can be marked as an IDP.  
However, this toxic mutant screen led to the serendipitous discovery of a silent mutation in _rpoZ_ (GCC to GCT at the 82 nd codon of _rpoZ_, frequent to a rare codon for alanine). As mentioned before, the silent mutant \(\omega\) has the same sequence of amino acids as that of wild-type protein, however, with a third base change in the code for alanine at the 82 nd position. The sequence of the mutant \(\omega\), having point mutation or silent in nature, was firmly established by mass spectroscopy-based sequence analysis [37]. The silent mutant has a folded conformation, despite having a wild-type sequence (\(\omega\)9, Figure 2A) and represents a classic case of two conformation of the same protein due to codon degeneracy. We have no experimental proof to pinpoint whether this is due to altered folding rate at  
Figure 2: (**A**) Far-UV CD (Far Ultra violet Circular Dichorism) spectra for _E. coli_\(\omega\) and its lethal mutants, \(\omega_{6}\), and \(\omega_{9}\). CD spectra for wild-type protein shows that wild-type \(\omega\) is less helical in comparison to structured mutants of \(\omega\). (**B**). The structure of \(\omega\)-subunit in RNAP assembly. The \(\omega\)-subunit exists as a well folded structure in the RNAP assembly in the crystal structure PDB id: 4 JKR [35], of _E. coli_ RNA polymerase.  
the RNA or protein level. However, the silent mutant _rpoZ_ or its cognate protein showed dominant lethal phenotype, expectedly. Following this observation, [PERSON] et al. generated many silent mutants to investigate the role of rare codon concerning its position in _rpoZ_ gene [34]. Out of those, a few mutants showed a lethal phenotype with concomitant structural alteration. However, most mutants did not show any difference in the growth profile or the secondary structure content. The authors observed that the mutants showing lethal phenotype were mainly mapped close to the C-terminal, falling in the protein's CR3 region. This region appears to be a sensitive region for mutation, as the other mutants isolated through toxic mutant screen were also mainly mapped in this region.