http://hdl.handle.net/2005/9 Sun, 09 Jun 2013 10:51:25 GMT 2013-06-09T10:51:25Z http://hdl.handle.net/2005/1957 Title: Evolutionary Design Of Active Site Plasticity In R.KpnI For Promiscuity In Metal Ion Utilization And Substrate Recognition Authors: Kommireddy, Vasu Abstract: Restriction modification (R-M) systems are important components of the prokaryotic arsenal against invading genomes. R-M systems directly target the foreign DNA and are often considered as primitive immune systems in bacteria. The defense system comprises of two contrasting enzymatic activities – a restriction endonuclease (REase) and a methyltransferase (MTase). Functionally, REases cleave a specific DNA sequence endonucleolytically at the phosphodiester bonds generating 5' or 3' overhangs or blunt ends. MTases catalyze the transfer of a methyl group from S-adenosyl-Lmethionine to adenine or cytosine. Four types of R–M systems are found in bacteria, viz., Types I, II, III and IV. Type II R-M systems, comprising of a separate REase and MTase, are the most abundant and well-studied enzymes. Type II REases recognize and cleave DNA within or near their recognition sequences. Surprisingly, these enzymes share little or no sequence homology amongst them. All the enzymes identified so far can be grouped into conventional PD-(D/E)XK, ββα-Me, GIY-YIG, phospholipase-derived and half-pipe endonucleases according to their folds and active site structures. Owing to their high specificity and defined cleavage pattern, they have become indispensable tools in molecular biology and have been widely exploited for studying protein–DNA interactions. The work presented in this thesis deals with R.KpnI, which belongs to the HNH superfamily of nucleases and is characterized by the presence of a ββα-Me finger motif. The REase isolated from Klebsiella pneumoniae recognizes the palindromic DNA sequence GGTAC/C and cleaves DNA as indicated. The enzyme is unique in exhibiting promiscuous DNA cleavage in the presence of Mg2+, a natural co-factor for a vast majority of REases. Surprisingly, Ca2+ and Zn2+ completely suppress the Mg2+ mediated promiscuous activity and induce high fidelity cleavage. These unusual features of R.KpnI led to the functional characterization of the ββα-Me finger active site motif. In addition, the studies were aimed at understanding the mechanism and the biological significance of substrate and co-factor promiscuity exhibited by the enzyme. The salient aspects of the thesis are summarized below. A general introduction and overview of the literature on structure-function studies, mechanism of recognition and catalysis by REases with special emphasis on Type II enzymes is presented in the Chapter 1. An account of co-factor specificity in REases, role of metal ions in DNA binding as well as in phosphodiester bond hydrolysis is provided. The various aspects of R-M systems that target the invading DNA elements and counter strategies employed by the foreign genomes to evade the restriction are also covered. The new developments that provide insights in understanding the diversity of R-M systems and additional biological roles that could increase the fitness of the host organism harboring them are described. The features of substrate and metal ion specificity in REases and the efforts undertaken to alter the specificity have been dealt at the end of the chapter. From the structures of the several ββα-Me finger nucleases, the α-helix has been implicated in providing a structural scaffold for the correct juxtapositioning of the catalytic residues. However, no mutagenesis data exists to delineate its role. Homology modeling studies of R.KpnI suggested a crossover structure for the α-helix of the ββαMe finger active site motif, which could possibly form dimeric interface and/or structural scaffold for the active site. Chapter 2 describes the computational modeling and mutational analysis performed to understand the role of the residues present in this α-helix in intersubunit interactions and/or stabilization of the active site. Mutation of the residues present in the α-helix lead to the loss of the enzyme activity, but not dimerization ability. Subsequent biophysical experiments showed that the α-helix of the ββα-Me finger of R.KpnI plays an important role for the stability of the protein–DNA complex needed for its function. In Chapter 3, unusual co-factor flexibility for R.KpnI is shown by using a battery of divalent metal co-factors differing in ionic radii and coordination geometries. A number of alkaline earth and transition group metal ions function as co-factors for DNA cleavage. The metal ions replaced each other readily from the enzyme’s active site revealing the active site plasticity. Mutation of the invariant His residue of the HNH motif caused abolition of the enzyme activity with all the co-factors indicating that the enzyme follows single metal ion mechanism for DNA cleavage. The indispensability of the invariant His in nucleophile activation together with the broad co-factor tolerance of the enzyme indicated the role of metal ions in electrostatic stabilization during catalysis. At higher concentrations, Mg2+, Mn2+ or Co2+ stimulate promiscuous cleavage while Cd2+, Ni2+ or Zn2+ inhibit phosphodiester bond hydrolysis. The underlying molecular mechanisms for the modulation of the enzyme activity by the metal ion binding to the second site are presented. Regulation of the endonuclease activity and fidelity by a second metal ion binding is a unique feature of R.KpnI among REases and HNH nucleases. The identification of additional metal ion binding residues would help in engineering REase variants with enhanced activity and/or specificity. Chapter 4 describes the generation of an R.KpnI variant with altered co-factor specificity by exploiting the active site plasticity of the enzyme. The mutant enzyme is a Mn2+ -dependent endonuclease defective in DNA cleavage with Mg2+ and other divalent metal ions. In the engineered mutant, only Mn2+ is selectively bound at the active site, imparting in vitro activity while being dormant in vivo. In addition to the Mn2+ selectivity, the mutant is impaired in concerted double-stranded DNA cleavage leading to the accumulation of nicked intermediates. The nicking activity of the mutant enzyme is further enhanced by altering the reaction conditions. Thus, a single point mutation in the active site of R.KpnI generates a Mn2+ -dependent REase and a sequence specific nicking endonuclease. The potential applications of such enzymes engineered for selective metal ion dependent activities have been discussed. R.KpnI is peculiar in retaining robust promiscuous cleavage despite being a typical Type II REase in all other characteristics. Chapter 5 presents results of the growth properties and phage titer analysis carried out with R.KpnI and its high fidelity variant to understand the biological significance of promiscuous activity. The enzyme isolated from the K. pneumoniae exhibited biochemical properties similar to that of R.KpnI overexpressed in E.coli. It was observed that the wild type but not the high fidelity variant could effectively restrict bacteriophages methylated at GGTACC. These results show that the REase exhibits promiscuous activity in vivo, which would be advantageous for the organism to better target the incoming foreign DNA. The promiscuous behavior of the R.KpnI could be one of the counter strategies employed by the bacteria against the constantly evolving phages in the co-evolutionary arms race. In conclusion, the work described in this thesis provides new insights about structure, function and biology of REases in general and R.KpnI in particular. The co-factor and substrate promiscuity of R.KpnI may indicate its evolutionarily intermediate form that is yet to attain a high degree of specificity. Alternatively, it is possible that this unique feature is retained during the evolution of the HNH REases serving some unknown function(s) in the cell, in addition to having an edge in countering the phage infections. Tue, 26 Mar 2013 18:30:00 GMT http://hdl.handle.net/2005/1957 2013-03-26T18:30:00Z http://hdl.handle.net/2005/1958 Title: Studies On The Structural And Biological Properties Of Rotavirus Enterotoxigenic Non-structural Protein 4 (NSP4) Authors: Palla, Narayan Sastri Abstract: Rotavirus is the major cause of infantile gastroenteritis. Each year more than 600,000 young children are estimated to die in developing countries throughout the world. Rotavirus infection can be either symptomatic or asymptomatic. But the genetic or molecular basis for rotavirus virulence is not yet clearly understood. NSP4, encoded by genome segment 10, is a multifunctional protein. It is identified as the first viral enterotoxin and is essential for virus morphogenesis and pathogenesis. Analysis of NSP4 from more than 175 strains failed to reveal any sequence motif or amino acid that segregated with the virulence phenotype of the virus. Further, a few studies indicated a lack of consistent correlation between virus virulence and diarrhea inducing ability of the cognate NSP4. To understand the basis for the inconsistency in the enterotoxigenic activity of a few NSP4s reported in a limited number of studies, comparative analysis of the biophysical, biochemical, and biological properties of NSP4ΔN72, which from SA11 and Hg18 was earlier shown to be highly diarrheagenic, from 17 different symptomatic and asymptomatic strains was carried out. To study structure-function relationship we used Thioflavin T fluorescence assay, gel filtration, CD spectroscopy, trypsin susceptibility and enterotoxin assay in newborn mice for all the proteins. Detailed comparative analysis of biochemical and biophysical properties and diarrheagenic activity of the recombinant ΔN72 peptides under identical conditions revealed wide differences among themselves in their resistance to trypsin cleavage, thoflavin T binding, multimerization and conformation without any correlation with their diarrhea inducing abilities. Since earlier studies showed that a secreted peptide (ΔN112) of SA11-NSP4 also induced diarrhea in newborn mice pups, we have generated NSP4ΔN112 deletions from six different strains and tested for their diarrhea inducing ability. The patterns of DD50 values of the ΔN112 peptides was similar to that for ΔN72 peptides, but were 1000-1200-fold less efficient than that of SA11ΔN72. NSP4 exists in multiple forms in the infected cells- as oligomers, higher molecular weight complexes and ER- and cytoplasmic membrane anchored forms. Previous studies suggest that the N-terminal boundary of the oligomerization domain could lie downstream to residue 94 from the N-terminus. A peptide from residue 112-175, secreted from rotavirus infected cells, was reported to induce dose-dependent diarrhea in suckling mice, suggesting that the N-terminal boundary of the enterotoxin activity could lie around residue 112. However, the precise N-terminal boundaries in NSP4 for oligomerization and diarrhea induction have not been identified. To address this question, a large number of deletion mutants C-terminal to residue 94 were generated and tested for their ability to induce diarrhea in newborn mouse pups. Our data suggest that while the deletions ∆N121 to ∆N131 failed to induce diarrhea, ΔN118 was diarrheagenic suggesting that the N-terminal boundary of the minimal diarrhea inducing domain lies between aa 118 and 121. Size exclusion chromatography revealed that residues 95 to 98 are critical and sufficient for oligomerization. Studies on oligomerization further revealed that NSP4ΔN94 exists in pentamers, tetramers and dimers, while deletion mutants C-terminal to aa 94 exist only as dimers. Our studies demonstrate for the first time that not only tetramers but pentamers as well as dimers possess enterotoxigenic properties. Most human rotavirus infections are caused by group A rotaviruses. Within this group, rotaviruses are further classified into subgroups based on the antigenic specificity associated with the protein product of the sixth gene, VP6. Previous studies have mapped SG I specificity to aa position 305 and the region between 296 and 299, and SG II specificity to residue 315 on VP6. However, the subgroup specific determinants on NSP4 have not been identified till date. In this study, we generated several amino acid substitution mutants in the SG I-specific SA11 NSP4∆N72 protein as in previous studies ∆N72 was found to efficiently bind DLPs. Using an enzyme linked immunosorbent assay method, the effect of the mutations in the C-terminal and N-terminal regions in ∆N72 on their binding ability to SG I and SG II DLPs was assayed. Residues at positions 85, 169, 174 and 175 and in the ISVD appear to collectively determine the specificity of binding to DLPs. While the conserved proline and glycines at positions 165, 168 and 162, respectively, are important for maintaining the required conformation for general recognition of DLP. The present study provides important insights towards understanding the determinants in NSP4 for SG-specific DLP interaction. Sun, 31 Mar 2013 18:30:00 GMT http://hdl.handle.net/2005/1958 2013-03-31T18:30:00Z http://hdl.handle.net/2005/2032 Title: Unravelling The Regulators Of Translation And Replication Of Hepatitis C Virus Authors: Ray, Upasana Abstract: Unravelling the regulators of translation and replication of Hepatitis C virus Hepatitis C virus (HCV) is a positive sense, single stranded RNA virus belonging to the genus Hepacivirus and the family Flaviviridae. It infects human liver cells predominantly. Although, the treatment with α interferon and ribavirin can control HCV in some cases, they fail to achieve sustained virological response in others, thus emphasizing the need of novel therapeutic targets. The viral genome is 9.6 kb long consisting of a 5’ untranslated region (5’UTR), a long open reading frame (ORF) that encodes the viral proteins and the 3’ untranslated region (3’UTR). The 5’UTR contains a cis acting element, the internal ribosome entry site (IRES) that mediates the internal initiation of translation. The HCV 5’UTR is highly structured and consists of four major stem-loops (SL) and a pseudoknot structure. HCV proteins are synthesized by the IRES mediated translation of the viral RNA, which is the initial obligatory step after infection. The viral proteins are synthesized in the form of a long continuous chain of proteins, the polyprotein, which is then processed by the host cell and the viral proteases. Once viral proteins are synthesized sufficiently, the viral RNA is replicated. However the mechanism of switch from translation to viral RNA replication is not well understood. Several host proteins as well as the viral proteins help in the completion of various steps in the HCV life cycle. In this thesis, the role of two such factors in HCV RNA translation and replication has been characterized and exploited to develop anti-HCV peptides. The HCV proteins are categorized into two major classes based on the functions broadly: the non structural and the structural proteins. HCV NS3 protein (one of the viral non structural proteins) plays a central role in viral polyprotein processing and RNA replication. In the first part of the thesis, it has been demonstrated that the NS3 protease (NS3pro) domain alone can specifically bind to HCV-IRES RNA, predominantly in the SLIV region. The cleavage activity of the NS3 protease domain is reduced upon HCV-RNA binding owing to the participation of the catalytic triad residue (Ser 139) in this RNA protein interaction. More importantly, NS3pro binding to the SLIV region hinders the interaction of La protein, a cellular IRES-trans acting factor required for HCV IRES-mediated translation, thus resulting in the inhibition of HCV-IRES activity. Moreover excess La protein could rescue the inhibition caused by the NS3 protease. Additionally it was observed that the NS3 protease and human La protein could out-compete each other for binding to the HCV SL IV region indicating that these two proteins share the binding region near the initiator AUG which was further confirmed using RNase T1 foot printing assay. Although an over expression of NS3pro as well as the full length NS3 protein decreased the level of HCV IRES mediated translation in the cells, replication of HCV RNA was enhanced significantly. These observations suggested that the NS3pro binding to HCV IRES reduces translation in favour of RNA replication. The competition between the host factor (La) and the viral protein (NS3) for binding to HCV IRES might contribute in the regulation of the molecular switch from translation to replication of HCV. In the second part the interaction of NS3 protease and HCV IRES has been elucidated in detail and the insights obtained were used to target HCV RNA function. Computational approach was used to predict the putative amino acid residues within the protease that might be involved in the interaction with the HCV IRES. Based on the predictions a 30-mer peptide (NS3proC-30) was designed from the RNA binding region. This peptide retained the RNA binding ability and also inhibited IRES mediated translation. The NS3proC-30 peptide was further shortened to 15-mer length (NS3proC-C15) and demonstrated ex vivo its ability to inhibit translation as well as replication. Additionally, its activity was tested in vivo in a mice model by encapsulating the peptide in Sendai virus based virosome followed by preferential delivery in mice liver. This virosome derived from Sendai virus F protein has terminal galactose moiety that interacts with the asialoglycoprotein receptor on the hepatocytes leading to membrane fusion and release of contents inside the cell. Results suggested that this peptide can be used as a potent anti-HCV agent. It has been shown earlier from our laboratory, that La protein interacts with HCVIRES near initiator AUG at GCAC motif by its central RNA recognition motif, the RRM2 (residues 112-184). A 24 mer peptide derived from this RRM2 of La (LaR2C) retained RNA binding ability and inhibited HCV RNA translation. NMR spectroscopy of the HCV-IRES bound peptide complex revealed putative contact points, mutations at which showed reduced RNA binding and translation inhibitory activity. The residues responsible for RNA recognition were found to form a turn in the RRM2 structure. A 7-mer peptide (LaR2C-N7) comprising this turn showed significant translation inhibitory activity. The bound structure of the peptide inferred from transferred NOE (Nuclear Overhauser Effect) experiments suggested it to be a βturn. Interestingly, addition of hexa-arginine tag enabled the peptide to enter Huh7 cells and showed inhibition HCV-IRES function. More importantly, the peptide significantly inhibited replication of HCVRNA. Smaller forms of this peptide however failed to show significant inhibition of HCV RNA functions suggesting that the 7-mer peptide as the smallest but efficient anti-HCV peptide from the second RNA recognition motif of the human La protein. Further, combinations of the LaR2C-N7 and NS3proC-C15 peptide showed better inhibitory activity. Both the peptides were found to be interacting at similar regions of SLIV around the initiator AUG. The two approaches have the potential to block the HCV RNA-directed translation by targeting the host factor and a viral protein, and thus can be tried in combination as a multi drug approach to combat HCV infection. Taken together, the study reveals important insights about the complex regulation of the HCV RNA translation and replication by the host protein La and viral NS3 protein. The interaction of the NS3 protein with the SLIV of HCV IRES leads to dislodging of the human La protein to inhibit the translation in favour of the RNA replication. These two proteins thus act as the regulators of the translation and the replication of viral RNA. The peptides derived from these regulators in turn regulate the functions of these proteins and inhibit the HCV RNA functions. Thu, 06 Jun 2013 18:30:00 GMT http://hdl.handle.net/2005/2032 2013-06-06T18:30:00Z http://hdl.handle.net/2005/1902 Title: Mechanism Of Activation Of Bacteriophage Mu Late Genes By Transcription Activator Protein C Authors: Swapna, Ganduri Abstract: Initiation of transcription is a major step in the regulation of gene expression. A dominant theme in regulation of gene expression lies in understanding the mechanism involved in selective expression of the genes in response to external or internal stimuli. Gene regulatory proteins bind DNA at specific sites either cognate to the promoters they act upon or at a distance, thereby exerting their effect by turning on (activation) or turning off (repression) the genes. Response of these factors to the environmental signals is further achieved by the DNA binding affinity of the transcription factors that can be modulated by small ligands, concentrations of which may fluctuate in response to nutrient availability and stress. Bacteriophages achieve a high degree of efficiency in gene expression by evolving elegant strategies of transcriptional control. mom gene of enterobacteriophage Mu serves as an excellent model to understand this elaborate regulation of gene expression. The gene encodes a unique DNA modification function that confers an anti-restriction phenotype to the phage genome. Though dispensable for phage growth, it is fascinating in two respects (i) a novel modification; (ii) regulation follows a complex scheme without precedence in prokaryotes. mom is the last gene to be expressed during the phage lytic life cycle. Premature expression of the gene is deleterious to both host and phage and hence it is under a complex regulatory network. Dam methylase, a host encoded protein acts as a positive regulator of gene expression, an example where methylation has been shown to play a positive role in regulating tranascription. OxyR, another host encoded protein negatively regulates mom gene expression. Dam methylation prevents the binding OxyR to its site located in the mom regulatory region. The regulatory interplay also involves two phage encoded proteins. C, a middle gene product is essential for transcriptional switch from middle to late genes and Com, a late gene product, for enhancing translation of mom mRNA. Thus, C and Com serve as transcriptional and translational activators of mom gene expression. Pmom is a weak promoter with both -10 and -35 elements away from consensus and a sub-optimal 19 bp spacer element encompassing a stretch of 6T residues that act as negative elements. ‘T stretch’ is known to induce a kink in the DNA. The sub-optimal spacer region makes the promoter elements out of phase and RNAP by itself cannot bind at mom promoter. C protein exerts its effect in activation in a multistep mechanism. The protein binds DNA as a dimer overlapping the promoter and unwinds the DNA, realigning the promoter elements, thus recruiting the RNAP. In the next step, it enhances the promoter clearance by the enzyme, thus enhancing the rate of productive transcription. With this prevailing knowledge on C mediated mom gene expression, the present thesis work describes the experiments carried out to further understand the molecular mechanism of second step activation at Pmom. Genetic and biochemical analysis were carried out to identify the interacting surface of C protein on RNAP. Subsequently, studies have been extended to understand the C mediated transactivation at other late promoters- lys, I, P, which encode for the lysis and morphogenetic functions of the phage. Finally, Mg2+ coordinating residues in C protein were identified to decipher the ligand induced conformational changes in the activator protein required for its transactivator function. Chapter I, a general introduction to the thesis, deals with the detailed discussion on gene expression and its regulatory mechanisms. RNA polymerase (RNAP) being the central molecule of gene expression (transcription) its organization and assembly are discussed. With the availability of the high resolution crystal structures of bacterial RNAP, an in-depth review on RNAP structure in terms of its potential regulatory targets, conformational changes associated with the formation of a functional holoenzyme, and during its transition from initiation to elongation processes have been described. Regulation of transcription with an emphasis on activation mechanism, ligand mediated allosteric transitions in regulatory proteins and the polymerase-activator interactions are discussed citing a few examples. The chapter concludes by introducing bacteriophage Mu and mom gene and its regulation by C. The objectives of the thesis form the concluding section of the chapter. Activators are capable of resurrecting defective promoters in response to cellular demands. The unusual, multistep activation of mom promoter (Pmom) by C protein involves activator mediated promoter unwinding to recruit RNA Polymerase (RNAP) and subsequent enhanced promoter clearance of the enzyme. The first step of transactivation is an interaction independent step, while the later might involve a transient interaction between C and one of the subunits of RNAP. Previous studies pointed out β′ subunit to be the most probable interaction partner. Chapter II comprises the genetic and biochemical studies carried out to confirm this observation. Employing a genetic screen mutations in rpoC gene (encoding the β′ subunit of RNAP), were isolated which result in the defective RNAP. The mutant RNAPs were assayed for their C specific activity by in vivo transactivation assays. Such mutants have been purified and characterized to understand their effect at different steps of C mediated mom gene expression during transcription initiation. The mutant RNAP had normal transcription activity with typical σ70 promoters but exhibited reduced productive transcription and enhanced abortive initiation on C-dependent Pmom. Experiments carried out to probe the interaction between C and mutant RNAP revealed that the physical interaction per se is not disrupted between the two proteins. Post C-mediated recruitment of RNAP to the promoter, transient interactions between the two proteins appears to induce subtle conformational changes in RNAP leading to an enhanced promoter clearance. Transactiavtor protein C is essential for the expression of other late genes lys, I, P apart from mom during the phage life cycle. Although the mechanism of multistep activation at Pmom has been elucidated, little is known on the transactivation from lys, I and P promoters. Chapter III includes studies carried out to understand the process of activation at these promoters. Owing to the differences in their C-binding site and promoter architecture it was important to investigate the differential effect of C, if any at lys, I , P promoters compared to that at Pmom. Activators in prokaryotes are shown to stimulate different steps of transcription initiation pathway ranging from the polymerase binding to the promoters to the post recruitment steps of isomerization and promoter clearance. Effect of C at different steps of transcription initiation pathway was analysed. The results indicate that C is absolutely essential for transcription from lys, I and P promoters similar to mom. However, at these promoters C exerts its effect at the step of Isomerisation from closed complex to open complex formation. Thus, C acts at a single step here and the mode of activation is different from that observed at Pmom. C dimer binds DNA with high affinity and sequence specificity, to an interrupted palindromic sequence overlapping the -35 element of mom promoter. Mg2+ mediated conformational transitions in C protein are essential for its DNA binding and transactivation functions. Chapter IV deals with the identification of the Mg2+ coordinating residues in C protein. Primary sequence analyses lead to the identification of a putative metal coordinating motif (EXDXD) towards the N-terminus of the protein. These residues were subjected to site directed mutagenesis to infer their role in Mg2+ coordination, its associated allosteric transition required for specific interaction with DNA. Mutants showed an altered Mg2+ induced conformation, compromised DNA binding and reduced levels of transcription activation when compared to C protein. Though Mg2+ is widely used in various DNA transaction reactions, this study provides the first insights on the importance of metal-ion induced allosteric transitions in regulating transcription factor function. Wed, 23 Jan 2013 18:30:00 GMT http://hdl.handle.net/2005/1902 2013-01-23T18:30:00Z