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Tagami S.,RIKEN | Tagami S.,Medical Research Council | Sekine S.-I.,RIKEN | Minakhin L.,Waksman Institute of Microbiology | And 7 more authors.
Genes and Development | Year: 2014

Transcription of DNA to RNA by DNA-dependent RNA polymerase (RNAP) is the first step of gene expression and a major regulation point. Bacteriophages hijack their host's transcription machinery and direct it to serve their needs. The gp39 protein encoded by Thermus thermophilus phage P23-45 binds the host's RNAP and inhibits transcription initiation from its major "-10/-35" class promoters. Phage promoters belonging to the minor "extended -10" class are minimally inhibited. We report the crystal structure of the T. thermophilus RNAP holoenzyme complexed with gp39, which explains the mechanism for RNAP promoter specificity switching. gp39 simultaneously binds to the RNAP β-flap domain and the C-terminal domain of the σ subunit (region 4 of the σ subunit [σ4]), thus relocating the β-flap tip and σ4. The ~45 Å displacement of σ4 is incompatible with its binding to the -35 promoter consensus element, thus accounting for the inhibition of transcription from -10/-35 class promoters. In contrast, this conformational change is compatible with the recognition of extended -10 class promoters. These results provide the structural bases for the conformational modulation of the host's RNAP promoter specificity to switch gene expression toward supporting phage development for gp39 and, potentially, other phage proteins, such as T4 AsiA. © 2014 Tagami et al.


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).


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.


Singaravelu G.,Waksman Institute of Microbiology | Chatterjee I.,Waksman Institute of Microbiology | Rahimi S.,Waksman Institute of Microbiology | Druzhinina M.K.,Waksman Institute of Microbiology | And 3 more authors.
Developmental Biology | Year: 2012

Despite undergoing normal development and acquiring normal morphology and motility, mutations in spe-38 or trp-3/spe-41 cause identical phenotypes in Caenorhabditis elegans-mutant sperm fail to fertilize oocytes despite direct contact. SPE-38 is a novel, four-pass transmembrane protein and TRP-3/SPE-41 is a Ca 2+-permeable channel. Localization of both of these proteins is confined to the membranous organelles (MOs) in undifferentiated spermatids. In mature spermatozoa, SPE-38 is localized to the pseudopod and TRP-3/SPE-41 is localized to the whole plasma membrane. Here we show that the dynamic redistribution of TRP-3/SPE-41 from MOs to the plasma membrane is dependent on SPE-38. In spe-38 mutant spermatozoa, TRP-3/SPE-41 is trapped within the MOs and fails to reach the cell surface despite MO fusion with the plasma membrane. Split-ubiquitin yeast-two-hybrid analyses revealed that the cell surface localization of TRP-3/SPE-41 is likely regulated by SPE-38 through a direct protein-protein interaction mechanism. We have identified sequences that influence the physical interaction between SPE-38 and TRP-3/SPE-41, and show that these sequences in SPE-38 are required for fertility in transgenic animals. Despite the mislocalization of TRP-3/SPE-41 in spe-38 mutant spermatozoa, ionomycin or thapsigargin induced influx of Ca 2+ remains unperturbed. This work reveals a new paradigm for the regulated surface localization of a Ca 2+-permeable channel. © 2012.


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.


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.


Nagornykh M.,Waksman Institute of Microbiology | Nagornykh M.,Russian Academy of Sciences | Zakharova M.,Russian Academy of Sciences | Protsenko A.,Russian Academy of Sciences | And 4 more authors.
Nucleic Acids Research | Year: 2011

The Eco29kI restriction-modification (R-M) system consists of two partially overlapping genes, eco29kIR, encoding a restriction endonuclease and eco29kIM, encoding methyltransferase. The two genes are thought to form an operon with the eco29kIR gene preceding the eco29kIM gene. Such an organization is expected to complicate establishment of plasmids containing this R-M system in naive hosts, since common logic dictates that methyltransferase should be synthesized first to protect the DNA from cleavage by the endonuclease. Here, we characterize the Eco29kI gene transcription. We show that a separate promoter located within the eco29kIR gene is sufficient to synthesize enough methyltransferase to completely modify host DNA. We further show that transcription from two intragenic antisense promoters strongly decreases the levels of eco29kIR gene transcripts. The antisense transcripts act by preventing translation initiation from the bicistronic eco29kIR-eco29kIM mRNA and causing its degradation. Both eco29kIM and antisense promoters are necessary for Eco29kI genes establishment and/or stable maintenance, indicating that they jointly contribute to coordinated expression of Eco29kI genes. © 2011 The Author(s).


PubMed | Waksman Institute of Microbiology and Rutgers University
Type: Journal Article | Journal: Plant physiology | Year: 2015

Cytoplasmic male-sterile (CMS) lines in maize (Zea mays) have been classified by their response to specific restorer genes into three categories: cms-C, cms-S, and cms-T. A mitochondrial genome representing each of the CMS cytotypes has been sequenced, and male sterility in the cms-S and cms-T cytotypes is linked to chimeric mitochondrial genes. To identify markers for plastid genotyping, we sequenced the plastid genomes of three fertile maize lines (B37, B73, and A188) and the B37 cms-C, cms-S, and cms-T cytoplasmic substitution lines. We found that the plastid genomes of B37 and B73 lines are identical. Furthermore, the fertile and CMS plastid genomes are conserved, differing only by zero to three single-nucleotide polymorphisms (SNPs) in coding regions and by eight to 22 SNPs and 10 to 21 short insertions/deletions in noncoding regions. To gain insight into the origin and transmission of the cms-T trait, we identified three SNPs unique to the cms-T plastids and tested the three diagnostic SNPs in 27 cms-T lines, representing the HA, I, Q, RS, and T male-sterile cytoplasms. We report that each of the tested 27 cms-T group accessions have the same three diagnostic plastid SNPs, indicating a single origin and maternal cotransmission of the cms-T mitochondria and plastids to the seed progeny. Our data exclude exceptional pollen transmission of organelles or multiple horizontal gene transfer events as the source of the mitochondrial urf13-T (unidentified reading frame encoding 13-kD cms-T protein) gene in the cms-T cytoplasms. Plastid genotyping enables a reassessment of the evolutionary relationships of cytoplasms in cultivated maize.


PubMed | Skolkovo Institute of Science and Technology, Russian Academy of Sciences, Waksman Institute of Microbiology, Purdue University and Saint Petersburg State Polytechnic University
Type: | Journal: Nucleic acids research | Year: 2017

CRISPR-Cas systems provide prokaryotes with adaptive defense against bacteriophage infections. Given an enormous variety of strategies used by phages to overcome their hosts, one can expect that the efficiency of protective action of CRISPR-Cas systems against different viruses should vary. Here, we created a collection of Escherichia coli strains with type I-E CRISPR-Cas system targeting various positions in the genomes of bacteriophages , T5, T7, T4 and R1-37 and investigated the ability of these strains to resist the infection and acquire additional CRISPR spacers from the infecting phage. We find that the efficiency of CRISPR-Cas targeting by the host is determined by phage life style, the positions of the targeted protospacer within the genome, and the state of phage DNA. The results also suggest that during infection by lytic phages that are susceptible to CRISPR interference, CRISPR-Cas does not act as a true immunity system that saves the infected cell but rather enforces an abortive infection pathway leading to infected cell death with no phage progeny release.


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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