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Batabyal S.,Sn Bose National Center For Basic Science | Mondol T.,Sn Bose National Center For Basic Science | Choudhury S.,Sn Bose National Center For Basic Science | Mazumder A.,Waksman Institute of Microbiology | Pal S.K.,Sn Bose National Center For Basic Science
Biochimie | Year: 2013

An overwhelming number of structural and functional studies on specific protein-DNA complexes reveal the existence of water molecules at the interaction interface. What role does the interfacial water molecules play in determining the specificity of association is thus a critical question. Herein, we have explored the dynamical role of minor groove water molecules and DNA side chain flexibility in lambda repressor-operator DNA interaction using well-characterized DNA minor groove binder dye, Hoechst 33258. The most striking finding of our studies reveals that the solvation time scale corresponding to the minor groove water molecules (∼50 ps) and DNA side chain flexibility (∼10 ns) remain unaltered even in protein-DNA complex in comparison to unbound operator DNA. The temperature dependent study further reveals the slower exchange of minor grove water molecules with bulk water in DNA-protein complex in comparison to the unbound DNA. Detailed structural studies including circular dichroism (CD) and Förster resonance energy transfer (FRET) have also been performed to elucidate the interaction between protein and DNA. © 2013 Elsevier Masson SAS. All rights reserved. Source

Restif C.,Rutgers University | Restif C.,Google | Ibanez-Ventoso C.,Rutgers University | Vora M.M.,Rutgers University | And 4 more authors.
PLoS Computational Biology | Year: 2014

In the effort to define genes and specific neuronal circuits that control behavior and plasticity, the capacity for high-precision automated analysis of behavior is essential. We report on comprehensive computer vision software for analysis of swimming locomotion of C. elegans, a simple animal model initially developed to facilitate elaboration of genetic influences on behavior. C. elegans swim test software CeleST tracks swimming of multiple animals, measures 10 novel parameters of swim behavior that can fully report dynamic changes in posture and speed, and generates data in several analysis formats, complete with statistics. Our measures of swim locomotion utilize a deformable model approach and a novel mathematical analysis of curvature maps that enable even irregular patterns and dynamic changes to be scored without need for thresholding or dropping outlier swimmers from study. Operation of CeleST is mostly automated and only requires minimal investigator interventions, such as the selection of videotaped swim trials and choice of data output format. Data can be analyzed from the level of the single animal to populations of thousands. We document how the CeleST program reveals unexpected preferences for specific swim "gaits" in wild-type C. elegans, uncovers previously unknown mutant phenotypes, efficiently tracks changes in aging populations, and distinguishes "graceful" from poor aging. The sensitivity, dynamic range, and comprehensive nature of CeleST measures elevate swim locomotion analysis to a new level of ease, economy, and detail that enables behavioral plasticity resulting from genetic, cellular, or experience manipulation to be analyzed in ways not previously possible. © 2014 Restif et al. Source

Gilmore J.M.,University of Georgia | Gilmore J.M.,University of Kansas | Gilmore J.M.,Waksman Institute of Microbiology | Bieber Urbauer R.J.,University of Georgia | And 7 more authors.
Biochemistry | Year: 2010

The AsiA protein is a T4 bacteriophage early gene product that regulates transcription of host and viral genes. Monomeric AsiA binds tightly to the - 70 subunit of Escherichia coli RNA polymerase, thereby inhibiting transcription from bacterial promoters and phage early promoters and coactivating transcription from phage middle promoters. Results of structural studies have identified amino acids at the protomer-protomer interface in dimeric AsiA and at the monomeric AsiA- 70 interface and demonstrated substantial overlap in the sets of residues that comprise each. Here we evaluate the contributions of individual interfacial amino acid side chains to protomer-protomer affinity in AsiA homodimers, to monomeric AsiA affinity for - 70, and to AsiA function in transcription. Sedimentation equilibrium, dynamic light scattering, electrophoretic mobility shift, and transcription activity measurements were used to assess affinity and function of site-specific AsiA mutants. Alanine substitutions for solvent-inaccessible residues positioned centrally in the protomer-protomer interface of the AsiA homodimer, V14, I17, and I40, resulted in the largest changes in free energy of dimer association, whereas alanine substitutions at other interfacial positions had little effect. These residues also contribute significantly to AsiA-dependent regulation of RNA polymerase activity, as do additional residues positioned at the periphery of the interface (K20 and F21). Notably, the relative contributions of a given amino acid side chain to RNA polymerase inhibition and activation (MotA-independent) by AsiA are very similar in most cases. The mainstay for intermolecular affinity and AsiA function appears to be I17. Our results define the core interfacial residues of AsiA, establish roles for many of the interfacial amino acids, are in agreement with the tenets underlying protein-protein interactions and interfaces, and will be beneficial for a general, comprehensive understanding of the mechanistic underpinnings of bacterial RNA polymerase regulation. © 2010 American Chemical Society. Source

Berdygulova Z.,Waksman Institute of Microbiology | Esyunina D.,Russian Academy of Sciences | Miropolskaya N.,Russian Academy of Sciences | Mukhamedyarov D.,Waksman Institute of Microbiology | And 6 more authors.
Nucleic Acids Research | Year: 2012

Gp39, a small protein encoded by Thermus thermophilus phage P23-45, specifically binds the host RNA polymerase (RNAP) and inhibits transcription initiation. Here, we demonstrate that gp39 also acts as an antiterminator during transcription through intrinsic terminators. The antitermination activity of gp39 relies on its ability to suppress transcription pausing at poly(U) tracks. Gp39 also accelerates transcription elongation by decreasing RNAP pausing and backtracking but does not significantly affect the rates of catalysis of individual reactions in the RNAP active center. We mapped the RNAP-gp39 interaction site to the β flap, a domain that forms a part of the RNA exit channel and is also a likely target for λ phage antiterminator proteins Q and N, and for bacterial elongation factor NusA. However, in contrast to Q and N, gp39 does not depend on NusA or other auxiliary factors for its activity. To our knowledge, gp39 is the first characterized phage-encoded transcription factor that affects every step of the transcription cycle and suppresses transcription termination through its antipausing activity. © 2012 The Author(s). Source

In a study published in Current Biology, Andrew Singson, a professor in the Department of Genetics in the School of Arts and Sciences, and colleagues from the National Institutes of Health and the College of William and Mary in Virginia, identified a protein, SPE-45, on the sperm of C. elegan worms that help bind sperm to eggs during fertilization. It is the same as the Izumo protein considered essential for humans and other mammals to reproduce that was discovered a decade ago by Japanese scientists who named it after a marriage shrine in Japan. "Humans and worms are connected by a common ancestor that lived more than 700 million years ago and this discovery will give us insight into their shared genetics and fertility pathways," said Singson, a principal investigator at the Waksman Institute of Microbiology. The research suggests that a common ancestor to both worms and humans had a SPE-45/Izumo-like gene that was required for sperm to function properly at fertilization, said Singson, who has been researching the biological process of fertility for the past two decades. "Twenty years ago when we started this research, we predicted that we would find the genes that are required for fertility from worms to humans," said Singson. "Now we know that this kind of molecule functions the same way beyond the mammalian branch of the tree of life." In the United States, one in eight couples has fertility problems. While about 70 percent of the cases can be attributed equally to the man or woman, 30 percent of the time no explanation can be found. In the new Rutgers study, scientists found that worms produced normal-looking sperm but could not create offspring because the sperm cell lacked the SPE-45 protein on its surface similar to sperm in humans and other mammals that lacked the Izumo protein. Blocking the protein prevents sperm from binding and fusing with the egg. "The protein works like molecular Velcro and helps the sperm and egg bind and fuse," said Singson. "This type of finding can play an indispensable role in understanding the biological process." The discovery was corroborated by a team of scientists working at Emory University in Georgia and Setsunan University in Japan. Taking a different approach and using computer analysis to look at DNA sequences, this international team came up with the same conclusion which was also published in Current Biology. Comparing the worm and mammalian DNA sequences they created a hybrid SPE-45/Izumo molecule that can cure infertility in worms. "This makes the results much more solid because two research groups have basically validated the results of the other," Signson said. Since studying human infertility directly is very challenging due to many ethical and experimental limitations, making a genetic connection between worms and humans will help in future treatments because scientists can do experiments in worms to learn more about the function of Izumo-like molecules that they cannot do in mammals, Singson said. "Finding new fertility genes in the worm can help us further understand the molecular basis of human fertility," he said. "The end result of this knowledge could be more informed and effective treatments for human infertility and reliable contraceptives for both sexes."

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