3. RNA polymerase (ββ’α 2 ω σ ) and its interactions at promoter. -35 (TTGACA), extended –10 (TGn) and -10 (TATAAT) elements are shown. (Adapted from Douglas F. Browning et al ., 2004) σ factors are essential for transcription initiation by virtue of their role in promoter recognition. Each of several sigma factors in cell is required for the transcription of a specific subset of genes operons. (Mooney., 2005).

6. RsbW: Anti-σ factor in B. subtilis stress response regulation (Adapted from Chien-Cheng et al ., 2003) RsbW is a kinase, and during normal exponential growth, RsbW inactivates RsbV by phosphorylation. RsbW is then free to bind σB and inhibit σB-dependent transcription. A drop in the levels of ATP forms one of the important stress signals and the cell responds by inhibiting RsbW activity by the dephosphorylation of RsbV and RsbV catalyzed release of σB from the RsbW-σB complex. (Alper et al., 1996) A separate regulatory mechanism allows induction of σB-dependent expression by stresses that have no direct effect on cell’s ATP levels. (Voelker et al., 1995, 1996)

8. Post-translational regulation of σ factor activity in Mycobacterium tuberculosis : Four anti- σ factors have been identified in the genome of M. tuberculosis ; RshA, RseA, RslA and UsfX. UsfX as the anti-σF factor debated since the identification of the σF operon. usfX (Rv3287c) is located upstream of sigF (Rv3286c) and the two are co-transcribed ( DeMaio et al ., 1997). UsfX has been found to inhibit σF -dependent transcription from σF promoter (Beaucher et al ., 2002). UsfX has been reported to be regulated by two antagonistic proteins: RsfA and RsfB. RsfA has been shown to respond to changes in redox potential, RsfB is believed to respond in phosphorylation dependent manner (Beaucher et al ., 2002). NO BIOCHEMICAL/STRUCTURAL INFORMATION ABOUT THESE PROTEINS: WHETHER UsfX HAS NUCLEOTIDE BINDING PROPERTIES AND KINASE PROPERTIES? IF ANY DO THEY HAVE A ROLE IN σF-UsfX INTERACTION? WHAT MAKES RsfB RESPOND TO CHANGES IN REDOX POTENTIAL AND HOW DOES THIS GOVERN UsfX-RsfA INTERACTION?

9. CHARACTERIZATION OF UsfX Data base searches: Tuberculist (http://genolist.pasteur.fr/Tuberculist/) database annotates Rv3287c as rsbW because of its homology to anti-sigma factor Regulator of SigB W of Bacillus subtilis . Later was named as UsfX (Upstream of SigF) for because of the identification of the Rv3287c as upstream of SigF with the initiation codon of SigF starting within UsfX (Beaucher et al ., 2002). Gene organisation in the SigF operon. (Adapted from http://genolist.pasteur.fr/TubercuList/)

10. Sequence Alignment Studies: Protein sequence homology searches of the UsfX using BLASTP against the available protein databases didn’t yield good scores for the alignment. Position specific iterated BLAST (PSI-BLAST) against the Protein Data Bank (PDB) and other data bases also didn’t provide good hits. PSI-BLAST against the non-redundant protein sequences (nr) database showed homologies with : COG 2172 (Anti-sigma regulatory factor, Serine/Threonine Protein Kinase). cd00075 (Histidine kinase-like ATPases). PRK 03660 (anti-sigma F factor) of Conserved Domain Database and similar clusters. Clusters Of Orthologous Groups (COGs) phylogenetically classifies proteins on the basis of the orthologous relations between them. COG2172 contains the cluster of anti-sigma regulatory factors/Serine-threonine protein kinases. A very low global sequence homology among the members from diverse backgrounds was observed. Mycobacteria UsfX sequences are well conserved with homology of up to 83 % for the respective proteins. Conclusively in spite of a low sequence homology UsfX is an anti-sigma factor with a common ancestral origin with the sequence characteristics conserved within the mycobacteria family.

11. Cloning, Over-expression and Purification

12. Nucleotide binding properties of UsfX Using Fluorophore labelled nucleotides didn’t give any significant results. Changes in the intrinsic trytophan fluorescence were monitored as function of nucleotide concentration. UsfX has a single tryptophan molecule: Trp-106. TITRATION CURVES

13. HILL PLOTS

14. The Kd values were determined from non-linear least-squares regression analysis of titration data using equation ΔF/ΔFmax=[Nucleotide]tot/(Kd +[ Nucleotide]tot ) The stoichiometry of binding was established from a linear version of the Hill equation, log(ΔF/ΔFmax–ΔF)=nlog[Nucleotide]-logK’ where n is the order of the binding reaction with respect to ligand concentration and K’is the concentration of nucleotide that yields 50% of ΔFmax. 0.97 1.6±0.1 205±5 1900±50 ADP 0.98 1.9±0.2 420±5 180±20 CTP 0.97 1.8±0.2 470±5 200±20 TTP 0.97 1.9±0.2 430±5 200±20 GTP 0.99 1.8±0.1 210±5 1300±50 ATP R 2 n H ΔF max(calc) K d (μM) Ligand

15. In silico analysis of nucleotide binding In silico modeling and docking approaches used to evaluate the binding of nucleotides. The binding site was identified based on a comparison with the B. stearothermophilus SpoIIAB co-ordinates . The nucleotide binding site of 1L0O is proximal to the UsfX Trp106 in the superposition (FIG A). Using Trp106 as the docking centre we confirmed tryptophan is a part of the nucleotide binding site. All the four nucleotides viz., ATP, GTP, CTP and TTP docked at the same site with the nucleotides interacting primarily through their ring moieties.

16. Nucleotide binding site is designed to accommodate a divalent ion also A D X S X S motif in the human integrin CR3 structure has been found to be involved in metal ion binding. Analysis of the UsfX model along with the sequence analysis lead to the identification of a conserved XGSFS motif in mycobacterial UsfX homologs where X is mainly a P or L.

17. 0.45 1 (γ quencher /γ acrylamide ) eff 3.2 10.94 11.3 17.3 K SV (eff) (M -1 ) UsfX Trp UsfX Trp Parameter I - Acrylamide Probing the nucleotide binding site with solvent quenchers confirmed that binding site has an affinity for positively charged molecules. ATP binding in presence and absence of MgCl 2 . MgCl 2 increases the binding of ATP as is evident from enhanced decrease in % Δ F. Ionic quenchers provide information about the polarity of the environment surrounding the tryptophan in proteins. 3.5 times decrease in accessibility towards KI.

18. Differential binding of adenosine nucleotides: Different ΔF max and K d values for a denosine nucleotides point towards differential behaviour in the binding site. GTP which is a pyrimidine like ATP has values similar to purines. Therefore the binding mode adapted by ATP is different as compared to the other nucleotides. K sv is the Stern-Volmer constant which depicts the accessibility of tryptophan molecule towards solvent. ΔF/F 0 is the fraction of fluorescence energy transfer from tryptophan to ligand. ASA is the accessible surface area around Trp-106 in free and bound states. Conclusively the binding of nucleotides in the active site is through their ring moieties but the significance of this differential binding of NTPs and differential response that this binding can produce may have an in vivo role which needs to be probed further. 53 0.98 0.87 3.58 GTP - 0.44 0.19 8.9 ADP 101 0.43 0.17 8.6 ATP 144 - - 9.53 NIL ASA (Å 2 ) around Trp-106 (ΔF/F 0 ) max (ΔF/F 0 ) 500μM K SV(500μM) (M -1 ) Ligand

19. NTPase activity of UsfX LEFT PANEL: ATPase activity (Above), GTPase activity (Below). RIGHT PANEL: ATPase assay in presence of unlabelled nucleotide competitors. Cold ATP was found to result in maximum decrease in the hydrolysis of the radio-labelled ATP. Both ATPase and GTPase activities found but ATPase activity almost 4 times GTPase activity. ATP is the preferred substrate inspite of GTP having higher affinity. No Kinase activity or auto-phosphorylation activity observed.

20. ANALYSIS OF UsfX- σ F INTERACTION Cloning, Over-expression and Purification of σ F Protein was over-expressed only when cells were induced at an OD 600 >2 probably due to the toxicity of the protein. Protein was soluble only in buffer containing β -mercaptoethanol. β -mercaptoethanol produced reduced state of cysteine residue which would otherwise form intermolecular disulfide linkages giving rise to large aggregates.

22. In the gel filtration analysis the elution volume of the complex pointed towards a molecular weight of 67kDa for the complex which is 7kDa more that when we presume complex to be formed by the interaction of a dimer of UsfX with a monomer of SigF. From densitometric analysis the proteins were estimated to bind in a ratio of 1.75:1 for UsfX/SigF. Hence UsfX and SigF bind in a stoichiometric ratio of 2:1. 1.75/1 Complex 2.24 19 16 15.6kDa UsfX 1.28 21 16 28.76kDa SigF Calculated stoichiometry Observed Intensity/mass Observed Intensity Monomeric mass Protein

23. The presence or absence of nucleotide did not have an apparent effect on the UsfX-SigF interaction so we probed the effect of SigF on UsfX-nucleotide interaction. Similar binding affinities for ATP and ADP confirms that the presence of a nucleotide in the nucleotide binding pocket of UsfX is not essential for UsfX-SigF interaction. There may be other in vivo factors essential for the formation of the UsfX-SigF complex. No significant change was observed in the nucleotide binding properties of UsfX which suggests that they bind with nearly same affinities in the apo UsfX and UsfX-SigF complex. The identical values of K d and ΔF max for UsfX and UsfX-SigF complex show the non-interference of the two ligands in binding of each other and makes us to believe that the location of the nucleotide binding site is apparently distal to the protein-protein interaction interface. Nucleotide binding properties of UsfX-SigF complex 215±10 205±10 1550±50 1900± 50 ADP 220±10 210±10 1250±50 1300± 50 ATP UsfX-SigF Complex UsfX UsfX-SigF Complex UsfX ΔF max(calc) K d (μM) Ligand

25. Sequence co-variation in RsfA homologs Proteins performing similar functions usually possess similar structural features like topologies and folds. The sequence perturbations in the primary sequence in one part of the structure mostly leads to changes in the other parts to accommodate/compensate for these changes. The co-variation in the residues of the protein sequence allows for the maintenance of the overall structural integrity. Scoring of sequence alignment : Positional entropy or informational entropy gives estimates about scores of multiple sequence alignment where the calculated values are normalized for the Shannon’s entropy so that conserved sequences i.e , those having low entropy score 1 and divergent sequences i.e , a high entropy score 0. Positional entropies were calculated for the aligned sequences of anti-anti sigma factors from the whole set retrieved from the database searches and for those sequences retrieved from mycobacterial sources. The analysis was carried out using the following criteria: (i) Overall positional entropy of 1 i.e ., a common residue in all samples. (ii) Positional entropy of 1 with respect to cysteine or serine containing proteins but overall entropy less than 1 i.e ., representative of the groups at that particular position or a primary co-variant signifying that the variation has come along with the change in the primary active residue. (iii) Positional entropy of 1 with respect to one group but less than 1 for the other group at that position i.e ., a secondary co-variant for that particular group.

26. Evolution of this class of anti-anti sigma factors has been accompanied by changes in the primary sequences which may be important for their functions under stress conditions. Group 1 describes the conserved residue in all the anti-anti sigma factor sequences. Group 2 can be described as the signature residues for their groups. Cys73 and Val63 of RsfA have replaced Ser61and Ile51 of RsfB respectively. The positional entropy at these positions within the group is 1 i.e ., the position is highly conserved over the whole sequence set. Group 3 describes the co-variations that have been brought by the introduction of cysteines in the sequence. The positions do not follow a conservation pattern in serine containing anti-anti sigma factors but does so in cysteine containing anti-sigma antagonists. So we can call them ‘structural co-variants’ . Cys109 is an example of ‘functional co-variant’ . It has to be essentially cysteine in redox sensor anti-anti sigma as it is the key to the formation of disulphide bond but is not conserved in serine containing antagonists.

27. Cloning, Over-expression and Purification of RsfA BL21(DE3) Origami host cells were essential for protein solubility potentially due to the disulfide bond of RsfA as Origami strain is important for maintaining the disulphide bonds.

29. Reduction of the Cys73-Cys109 increases the hydrodynamic radius of the protein. Elution volume of the native protein corresponds to a Stokes radius (R S ) of 18.5Å while that of the disulphide bond reduced form is 22.1Å. Reduction of the disulphide bond might result in increased flexibility of the protein and allow for the enhanced hydrodynamic radius which might be necessary for interaction with UsfX. Effect of DTT on conformation of RsfA DTT is a reducing agent and mimics the increased reduced environment that cells encounter in the stress phase. RsfA binds to UsfX only when the disulphide bond is reduced. However, the actual significance of the disulphide bond in regulating its interactions with UsfX is not known.

30. Upper panel : Residues around Cys109 of RsfA where the Cys73-Cys109 disulphide bridge is maintained. The snapshot is at the beginning of the MD simulations (Left) and at the end of the MD run (Right). Lower panel: Snapshot of the same region at the beginning of the MD simulations where the Cys73-Cys109 disulphide bridge is broken ( Left ) and after the MD simulations. Rearrangement of the His107 and Phe104 side chains of UsfX takes place where the side chain of His107 comes close to Cys109 of RsfA. Interactions at the UsfX-RsfA interface

31. Conclusively, A sigma factor (SigF), an anti-sigma factor (UsfX) and an anti-anti-sigma factor (RsfA) from M. tuberculosis H37Rv were cloned, over-expressed and purified in E. coli based expression systems. The anti-sigma factor, UsfX is a dimeric protein in solution with the monomeric molecular weight of about 15kDa. Two independent nucleotide binding sites were identified to be present in the UsfX dimer. Binds all the naturally occurring nucleotides which has not been reported from any other source. Adenosine nucleotides behave in a different manner in the binding pocket. May be has an in vivo role in transcription regulation? The nucleotide binding pocket like other nucleotide binding proteins is designed to accommodate divalent ions which enhances the binding of nucleotides. Although the nucleotide binding properties are like homologous proteins but the residues involved in the binding site are different from known proteins. The sequence is highly conserved within the mycobacteria family. An XGSFS motif can be identified in mycobacterial UsfX homologs which along with tryptophan form the core of the binding pocket. No kinase or autophosphorylation activity was observed in UsfX. But weak NTPase activity was observed with preference for ATP as the substrate.

32. The sigma factor SigF is a monomer is solution with a molecular weight of about 28kDa. SigF-UsfX complex was co-purified using a co-expression vector system. No in vitro interaction could be observed between the two proteins even in the presence of the nucleotides. Some unknown factors/conditions apparently support the interaction. The two ligands: nucleotide and SigF didn’t produce any observable effect on the binding of one another. Thus binding of SigF to UsfX is independent of nucleotide binding. The anti-anti-sigma factor RsfA is a monomer is solution with a molecular weight of about 28kDa. Sequence analysis of RsfA points towards the structural and functional co-varience of the residues along with the functionally important Cys-Cys disulfide bridge in this mycobacterial specific class of anti-sigma factor antagonists. The Cys-Cys disulfide bridge serves a dual role: Acts as a sensor for the increased redox potential in the Stress phase and the reduction of this bond facilitates its interaction with UsfX which renders SigF free. Published results: M. tuberculosis UsfX (Rv3287c) exhibits novel nucleotide binding and hydrolysis properties. Biochemical and Biophysical Research Communications 375 (2008) 465–470. Interactions of the M. tuberculosis UsfX with the cognate sigma factor SigF and anti-anti-sigma factor RsfA. Biochimica et Biophysica Acta 1794 (2009) 541–553.

34. Acknowledgement Dr. R. Ravishankar (My Ph.D mentor). Dr. Amoghanant Sahasrabudhe (CDRI). Dr. Shantunu Chaudhary (IGIB, New Delhi). Dr. J.V.Pratap. All the members of my own lab, Dr.J.V.Pratap’s group, MSB Division and CDRI fraternity. Special thanks to Dr. Sandeep K. Srivastava.

35. THANKS