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The SmtB/ArsR family of prokaryotic metalloregulators are winged-helix transcriptional repressors that collectively provide resistance to a wide range of both biologically required and toxic heavy metal ions. CmtRMtb is a recently described CdII/PbII regulator expressed in M. tuberculosis that is structurally distinct from the wellcharacterized SmtB/ArsR CdII/PbII sensor, S. aureus plasmid pI258-encoded CadC. From functional analyses and a multiple sequence alignment of CmtR homologs, CmtRMtb is proposed to bind PbII and CdII via coordination by Cys57, Cys61 and Cys102 [Cavet et al. (2003) J. Biol. Chem. 278, 44560-44566]. To better understand the mechanism how CmtRMtb utilizes specific metal ions to perform transcriptional repressor function, both CmtRMtb and its homolog in S. coelicolor A3(2) (CmtRSc) were studied. We establish here that both wild-type and C102S CmtRMtb are homodimers and bind CdII and PbII via formation of cysteine thiolate-rich coordination bonds. UV-Vis optical spectroscopy and 113Cd NMR spectroscopy (δ=480 ppm) suggest two or three thiolate donors, while 111mCd perturbed angular correlation (PAC) spectroscopy establish an unusual trigonal pyramidal coordination eometry. C102S CmtRMtb binds CdII and ZnII with only ≈ 10-20 fold lower affinity relative to wild-type CmtRMtb, but ≈ 100-1000-fold lower for PbII. Quantitative investigation of CmtR-cmt O/P binding equilibria using fluorescence anisotropy reveals that Cys57 and Cys61 anchor the coordination complex with Cys102 functioning as a key allosteric ligand, while playing only an accessory role in stabilizing the metal complex in the free protein. Similar metal titration experiments were carried out with a putative CmtR homolog from S. coelicolor A3(2) (CmtRSc) and a double cysteine substitution mutant C110G/C111S CmtRSc. The implications of these findings on the evolution of distinct metal sensing sites in a family of homologous proteins are discussed.

The biological activities of retinoic acid (RA) and its synthetic analogues are mediated through nuclear receptors, termed retinoic acid receptors (RARs) and retinoid X receptors (RXRs). The transcriptional activity of RAR on target gene expression is achieved by its AF-1 domain and AF-2 domain. The function of AF-2 is known to be mediated by a number of coregulatory proteins. However the mechanism of AF-1 function is not well studied. We have hypothesized that the AF-1 function of RAR is regulated by specific interacting proteins. HACE1 was identified as an AF-1 domain interacting protein in a yeast two-hybrid screen. HACE1 interacts with RAR&beta<sub>3 / Microbiology and Immunology

This thesis describes the isolation and characterisation of the C. albicans NRG1 gene, which encodes a repressor of filamentous growth in this pathogenic fiingus. A C. albicans SBP1 cDNA was previously isolated in a screen for transacting factors that bind to a STRE-like element (consensus sequence: CCCCT) (Leng, 1999). In S. cerevisiae, STRE is a stress-responsive element that is required for the regulation of many stress-responsive genes (Marchler et al., 1993). In C. albicans, this element had been identified in the promoters of two hypha-specific genes, ALS8 and HYR1. Since many conditions that induce yeast-hypha morphogenesis in C. albicans impose a stress, it was proposed that the STRE- binding protein (Sbpl) might influence yeast-hypha morphogenesis and/or stress responses in this human pathogen. The cDNA was then used to isolate the complete C. albicans SBP1 locus by colony hybridisation. Both the cDNA and gene were sequenced, revealing an ORF capable of encoding a protein of 310 amino acids containing a C2H2-zinc finger motifs near its C-terminus. The zinc finger region of this protein displayed the highest sequence similarity to S. cerevisiae NRG1 (67 % identity), and hence the gene was renamed CaNRGl. To examine the role of CaNrgl, a C. albicans nrgl/nrgl null mutant and a mutant over-expressing the NRG1 gene were created. Overexpression of NRG1 did not reveal any obvious phenotypes, but inactivation of NRG1 caused constitutive filamentous and invasive growth, as well as increased sensitivity to some stresses. Also, the expression of the hypha-specific genes, ALS8, ECE1, HWP1 and HYR1, was derepressed in the nrgl/nrgl mutants. Similar phenotypes were observed for a C. albicans tupl/tupl null mutant. These observations suggest that Nrgl represses filamentous growth in C. albicans, possibly by recruiting Tupl to specific promoters. Unlike the tupl/tupl mutant, nrgl/nrgl cells formed normal hyphae following pH and serum stimulation, they generated chlamydospores at normal rates, and they grew at 42 C. Transcript profiling of 2002 C. albicans genes revealed that Nrgl regulates a subset of Tupl-repressed genes, which includes known hypha- specific genes and some virulence factors. The data also showed that Tupl regulates other genes, which are not regulated by Nrgl, including glucose sensitive genes, amino acid and sterol biosynthesis genes, and genes encoding other virulence determinants. Taken together, this study demonstrates that Nrgl is a transcriptional repressor that regulates a set of functions required for yeast-hypha morphogenesis and virulence in C. albicans.

abstract: The highly predictable structural and thermodynamic behavior of deoxynucleic acid (DNA) and ribonucleic acid (RNA) have made them versatile tools for creating artificial nanostructures over broad range. Moreover, DNA and RNA are able to interact with biological ligand as either synthetic aptamers or natural components, conferring direct biological functions to the nucleic acid devices. The applications of nucleic acids greatly relies on the bio-reactivity and specificity when applied to highly complexed biological systems. This dissertation aims to 1) develop new strategy to identify high affinity nucleic acid aptamers against biological ligand; and 2) explore highly orthogonal RNA riboregulators in vivo for constructing multi-input gene circuits with NOT logic. With the aid of a DNA nanoscaffold, pairs of hetero-bivalent aptamers for human alpha thrombin were identified with ultra-high binding affinity in femtomolar range with displaying potent biological modulations for the enzyme activity. The newly identified bivalent aptamers enriched the aptamer tool box for future therapeutic applications in hemostasis, and also the strategy can be potentially developed for other target molecules. Secondly, by employing a three-way junction structure in the riboregulator structure through de-novo design, we identified a family of high-performance RNA-sensing translational repressors that down-regulates gene translation in response to cognate RNAs with remarkable dynamic range and orthogonality. Harnessing the 3WJ repressors as modular parts, we integrate them into biological circuits that execute universal NAND and NOR logic with up to four independent RNA inputs in Escherichia coli. / Dissertation/Thesis / Doctoral Dissertation Biochemistry 2019

<i>Escherichia coli </i>RecA is a versatile protein that is involved in homologous recombination, and coordination of both the DNA damage response and translesion synthesis. Single-stranded DNA (ssDNA) that is generated at the site of double-stranded breaks serves as a signal to activate RecA. This allows RecA to form a long helical filament on the ssDNA, which is required in recombination, hydrolysis of ATP, and mediating the self-cleavage of some ser-lys dyad proteins such as the LexA repressor. In this thesis, the formation of the RecA/LexA complex did not require preactivation by ssDNA, instead a volume excluding agent in the presence of LexA was able to stimulate its formation. These preliminary results led to a hypothesis that the formation of the RecA/LexA complex is a thermodynamic process that involves three steps: (1) a change in RecAs conformation towards the active form, (2) a change in LexAs conformation towards the cleavable form (i.e. burial of the ser-lys dyad catalytic residues), and (3) the binding between the active form of RecA and the cleavable form of LexA. Evidence for this model was shown by the ability of either NaCl, LexA K156A, an ATP substrate, or a volume excluding agent to enhance the stability of the RecA/LexA complex, which was detected by both the ATPase and coprotease assays. Hyper-active RecA mutants, isolated form the yeast two-hybrid screen, were also tested, however they did not enhance the stability of the complex. Additionally, RecAs binding preference for the monomer or dimer form of LexA was examined, since it is unknown which species of LexA is able to enhance the stability of the complex. To generate the monomer form of LexA, single point mutations were introduced at the dimer interface of the protein such that its dimerization was disrupted by charge-charge repulsions. Based on the inhibition assay, RecA was found to bind preferentially to dimer form and not the monomer form of LexA, possible reasons for these results are discussed.

<i>Escherichia coli </i>RecA is a versatile protein that is involved in homologous recombination, and coordination of both the DNA damage response and translesion synthesis. Single-stranded DNA (ssDNA) that is generated at the site of double-stranded breaks serves as a signal to activate RecA. This allows RecA to form a long helical filament on the ssDNA, which is required in recombination, hydrolysis of ATP, and mediating the self-cleavage of some ser-lys dyad proteins such as the LexA repressor. In this thesis, the formation of the RecA/LexA complex did not require preactivation by ssDNA, instead a volume excluding agent in the presence of LexA was able to stimulate its formation. These preliminary results led to a hypothesis that the formation of the RecA/LexA complex is a thermodynamic process that involves three steps: (1) a change in RecAs conformation towards the active form, (2) a change in LexAs conformation towards the cleavable form (i.e. burial of the ser-lys dyad catalytic residues), and (3) the binding between the active form of RecA and the cleavable form of LexA. Evidence for this model was shown by the ability of either NaCl, LexA K156A, an ATP substrate, or a volume excluding agent to enhance the stability of the RecA/LexA complex, which was detected by both the ATPase and coprotease assays. Hyper-active RecA mutants, isolated form the yeast two-hybrid screen, were also tested, however they did not enhance the stability of the complex. Additionally, RecAs binding preference for the monomer or dimer form of LexA was examined, since it is unknown which species of LexA is able to enhance the stability of the complex. To generate the monomer form of LexA, single point mutations were introduced at the dimer interface of the protein such that its dimerization was disrupted by charge-charge repulsions. Based on the inhibition assay, RecA was found to bind preferentially to dimer form and not the monomer form of LexA, possible reasons for these results are discussed.

The repression of the E. coli lac operon seems to be more efficient than the current theoretical model allows for. Specifically, it is more quiet than expected during the replication of the chromosome. I have induced cells during short periods and counted the number of protein products from the operon to determine if there is a delay in activation of transcription that could account for the discrepancy. The results are compatible with a delay of 10-20 s, but the delay could not be conclusively proven. Furthermore, it has been investigated if the mechanism behind the delay might be differential localization of the lac operon with and without induction. It is shown that the lac operon is more often located in the periphery of the cell and in the internucleoid region when induced. These might be regions where genes are higher expressed, giving a delay in expression after de-repression before the gene is transported there.

Diss. (sammanfattning) Uppsala : Univ., 2004. / Härtill 3 uppsatser.

abstract: Molecular dynamics (MD) simulations provide a particularly useful approach to understanding conformational change in biomolecular systems. MD simulations provide an atomistic, physics-based description of the motions accessible to biomolecular systems on the pico- to micro-second timescale, yielding important insight into the free energy of the system, the dynamical stability of contacts and the role of correlated motions in directing the motions of the system. In this thesis, I use molecular dynamics simulations to provide molecular mechanisms that rationalize structural, thermodynamic, and mutation data on the interactions between the lac repressor headpiece and its O1 operator DNA as well as the ERK2 protein kinase. I performed molecular dynamics simulations of the lac repressor headpiece - O1 operator complex at the natural angle as well as at under- and overbent angles to assess the factors that determine the natural DNA bending angle. I find both energetic and entropic factors contribute to recognition of the natural angle. At the natural angle the energy of the system is minimized by optimization of protein-DNA contacts and the entropy of the system is maximized by release of water from the protein-DNA interface and decorrelation of protein motions. To identify the mechanism by which mutations lead to auto-activation of ERK2, I performed a series of molecular dynamics simulations of ERK1/2 in various stages of activation as well as the constitutively active Q103A, I84A, L73P and R65S ERK2 mutants. My simulations indicate the importance of domain closure for auto-activation and activity regulation. My results enable me to predict two loss-of-function mutants of ERK2, G83A and Q64C, that have been confirmed in experiments by collaborators. One of the powerful capabilities of MD simulations in biochemistry is the ability to find low free energy pathways that connect and explain disparate structural data on biomolecular systems. An extention of the targeted molecular dynamics technique using constraints on internal coordinates will be presented and evaluated. The method gives good results for the alanine dipeptide, but breaks down when applied to study conformational changes in GroEL and adenylate kinase. / Dissertation/Thesis / Ph.D. Chemistry 2011