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Vershon, AK, Pierce M.  2000.  Transcriptional Regulation of Meiosis in Yeast. Curr Opin Cell Biol. 12:334-339. Abstract
The genes required for meiosis and sporulation in yeast are expressed at specific points in a highly regulated temporal pathway. Recent experiments using DNA microarrays to examine gene expression during meiosis and the identification of many regulatory factors have provided important advances in our understanding of how genes are regulated at the different stages of meiosis.
Vieira, J, Messing J.  1982.  The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene. 19:259-68. AbstractWebsite
A series of plasmid vectors containing the multiple cloning site (MCS7) of M13mp7 has been constructed. In one of these vectors a kanamycin-resistance marker has been inserted into the center of the symmetrical MCS7 to yield a restriction-site-mobilizing element (RSM). The drug-resistance marker can be cleaved out of this vector with any of the restriction enzymes that recognize a site of the flanking sequences of the RSM to generate an RSM with either various sticky ends or blunt ends. These fragments can be used for insertion mutagenesis of any target molecule with compatible restriction sites. Insertion mutants are selected by their resistance to kanamycin. When the drug-resistance marker is removed with PstI, a small in-frame insertion can be generated. In addition, two new MCSs having single restriction sites have been formed by altering the symmetrical structure of MCS7. The resulting plasmids pUC8 and pUC9 allow one to clone doubly digested restriction fragments separately with both orientations in respect to the lac promoter. The terminal sequences of any DNA cloned in these plasmids can be characterized using the universal M13 primers.
Vinyard, DJ, Zachary CE, Ananyev GM, Dismukes CG.  2013.  Thermodynamically accurate modeling of the catalytic cycle of photosynthetic oxygen evolution: a mathematical solution to asymmetric Markov chains.. Biochimica et biophysica acta. 1827(7):861-8. Abstract
Forty-three years ago, Kok and coworkers introduced a phenomenological model describing period-four oscillations in O2 flash yields during photosynthetic water oxidation (WOC), which had been first reported by Joliot and coworkers. The original two-parameter Kok model was subsequently extended in its level of complexity to better simulate diverse data sets, including intact cells and isolated PSII-WOCs, but at the expense of introducing physically unrealistic assumptions necessary to enable numerical solutions. To date, analytical solutions have been found only for symmetric Kok models (inefficiencies are equally probable for all intermediates, called "S-states"). However, it is widely accepted that S-state reaction steps are not identical and some are not reversible (by thermodynamic restraints) thereby causing asymmetric cycles. We have developed a mathematically more rigorous foundation that eliminates unphysical assumptions known to be in conflict with experiments and adopts a new experimental constraint on solutions. This new algorithm termed STEAMM for S-state Transition Eigenvalues of Asymmetric Markov Models enables solutions to models having fewer adjustable parameters and uses automated fitting to experimental data sets, yielding higher accuracy and precision than the classic Kok or extended Kok models. This new tool provides a general mathematical framework for analyzing damped oscillations arising from any cycle period using any appropriate Markov model, regardless of symmetry. We illustrate applications of STEAMM that better describe the intrinsic inefficiencies for photon-to-charge conversion within PSII-WOCs that are responsible for damped period-four and period-two oscillations of flash O2 yields across diverse species, while using simpler Markov models free from unrealistic assumptions.
Vinyard, DJ, Sun JS, Gimpel J, Ananyev GM, Mayfield SP, Dismukes GC.  2016.  Natural isoforms of the Photosystem II D1 subunit differ in photoassembly efficiency of the water-oxidizing complex.. Photosynth Res.. Abstract
Oxygenic photosynthesis efficiency at increasing solar flux is limited by light-induced damage (photoinhibition) of Photosystem II (PSII), primarily targeting the D1 reaction center subunit. Some cyanobacteria contain two natural isoforms of D1 that function better under low light (D1:1) or high light (D1:2). Herein, rates and yields of photoassembly of the Mn4CaO5 water-oxidizing complex (WOC) from the free inorganic cofactors (Mn2+, Ca2+, water, electron acceptor) and apo-WOC-PSII are shown to differ significantly: D1:1 apo-WOC-PSII exhibits a 2.3-fold faster rate-limiting step of photoassembly and up to seven-fold faster rate to the first light-stable Mn3+ intermediate, IM1*, but with a much higher rate of photoinhibition than D1:2. Conversely, D1:2 apo-WOC-PSII assembles slower but has up to seven-fold higher yield, achieved by a higher quantum yield of charge separation and slower photoinhibition rate. These results confirm and extend previous observations of the two holoenzymes: D1:2-PSII has a greater quantum yield of primary charge separation, faster [P680 + Q A - ] charge recombination and less photoinhibition that results in a slower rate and higher yield of photoassembly of its apo-WOC-PSII complex. In contrast, D1:1-PSII has a lower quantum yield of primary charge separation, a slower [P680 + Q A - ] charge recombination rate, and faster photoinhibition that together result in higher rate but lower yield of photoassembly at higher light intensities. Cyanobacterial PSII reaction centers that contain the high- and low-light D1 isoforms can tailor performance to optimize photosynthesis at varying light conditions, with similar consequences on their photoassembly kinetics and yield. These different efficiencies of photoassembly versus photoinhibition impose differential costs for biosynthesis as a function of light intensity.
Vinyard, DJ, Xu Y, Bennette N, McNeely K, Bryant DA, Dismukes CG.  2013.  Natural osmolytes are much less effective substrates than glycogen for catabolic energy production in the marine cyanobacterium Synechococcus sp. strain PCC 7002.. Journal of biotechnology. 166(3):65-75. Abstract
ADP-glucose pyrophosphorylase, encoded by glgC, catalyzes the first step of glycogen and glucosylglycer(ol/ate) biosynthesis. Here we report the construction of the first glgC null mutant of a marine cyanobacterium (Synechococcus sp. PCC 7002) and investigate its impact on dark anoxic metabolism (autofermentation). The glgC mutant had 98% lower ADP-glucose, synthesized no glycogen and produced appreciably more soluble sugars (mainly sucrose) than wild type (WT). Some glucosylglycerol was still observed, which suggests that the mutant has another, inefficient ADP-glucose synthesis pathway. In contrast, hypersaline conditions (1M NaCl) were lethal to the mutant strain, indicating that, unlike other strains, the elevated sucrose does not compensate for the reduced GG as osmolyte. In contrast to WT, nitrate limitation did not cause bleaching of N-containing pigments or carbohydrate accumulation in the glgC mutant, indicating impaired recycling of nitrogen stores. Despite the 2-fold increase in osmolytes, both the respiration and autofermentation rates of the glgC mutant were appreciably slower (2-4-fold) and correlated quantitatively with the lower fraction of insoluble carbohydrates relative to WT (85% vs. 12%). However, the remaining insoluble carbohydrates still accounted for a high fraction of the carbohydrate catabolized (38%), indicating that insoluble carbohydrates rather than osmolytes were the preferred substrate for autofermentation.
Vinyard, DJ, Ananyev GM, Dismukes CG.  2013.  Photosystem II: the reaction center of oxygenic photosynthesis.. Annual review of biochemistry. 82:577-606. Abstract
Photosystem II (PSII) uses light energy to split water into chemical products that power the planet. The stripped protons contribute to a membrane electrochemical potential before combining with the stripped electrons to make chemical bonds and releasing O2 for powering respiratory metabolisms. In this review, we provide an overview of the kinetics and thermodynamics of water oxidation that highlights the conserved performance of PSIIs across species. We discuss recent advances in our understanding of the site of water oxidation based upon the improved (1.9-Å resolution) atomic structure of the Mn4CaO5 water-oxidizing complex (WOC) within cyanobacterial PSII. We combine these insights with recent knowledge gained from studies of the biogenesis and assembly of the WOC (called photoassembly) to arrive at a proposed chemical mechanism for water oxidation.
Vinyard, DJ, Gimpel J, Ananyev GM, Mayfield SP, Dismukes CG.  2014.  Engineered Photosystem II reaction centers optimize photochemistry versus photoprotection at different solar intensities.. Journal of the American Chemical Society. 136(10):4048-55. Abstract
The D1 protein of Photosystem II (PSII) provides most of the ligating amino acid residues for the Mn4CaO5 water-oxidizing complex (WOC) and half of the reaction center cofactors, and it is present as two isoforms in the cyanobacterium Synechococcus elongatus PCC 7942. These isoforms, D1:1 and D1:2, confer functional advantages for photosynthetic growth at low and high light intensities, respectively. D1:1, D1:2, and seven point mutations in the D1:2 background that are native to D1:1 were expressed in the green alga Chlamydomonas reinhardtii. We used these nine strains to show that those strains that confer a higher yield of PSII charge separation under light-limiting conditions (where charge recombination is significant) have less efficient photochemical turnover, measured in terms of both a lower WOC turnover probability and a longer WOC cycle period. Conversely, these same strains under light saturation (where charge recombination does not compete) confer a correspondingly faster O2 evolution rate and greater protection against photoinhibition. Taken together, the data clearly establish that PSII primary charge separation is a trade-off between photochemical productivity (water oxidation and plastoquinone reduction) and charge recombination (photoprotection). These trade-offs add up to a significant growth advantage for the two natural isoforms. These insights provide fundamental design principles for engineering of PSII reaction centers with optimal photochemical efficiencies for growth at low versus high light intensities.
Vinyard, DJ, Gimpel J, Ananyev GM, Cornejo MA, Golden SS, Mayfield SP, Dismukes CG.  2013.  Natural variants of photosystem II subunit D1 tune photochemical fitness to solar intensity.. The Journal of biological chemistry. 288(8):5451-62. Abstract
Photosystem II (PSII) is composed of six core polypeptides that make up the minimal unit capable of performing the primary photochemistry of light-driven charge separation and water oxidation in all oxygenic phototrophs. The D1 subunit of this complex contains most of the ligating amino acid residues for the Mn(4)CaO(5) core of the water-oxidizing complex (WOC). Most cyanobacteria have 3-5 copies of the psbA gene coding for at least two isoforms of D1, whereas algae and plants have only one isoform. Synechococcus elongatus PCC 7942 contains two D1 isoforms; D1:1 is expressed under low light conditions, and D1:2 is up-regulated in high light or stress conditions. Using a heterologous psbA expression system in the green alga Chlamydomonas reinhardtii, we have measured growth rate, WOC cycle efficiency, and O(2) yield as a function of D1:1, D1:2, or the native algal D1 isoform. D1:1-PSII cells outcompete D1:2-PSII cells and accumulate more biomass in light-limiting conditions. However, D1:2-PSII cells easily outcompete D1:1-PSII cells at high light intensities. The native C. reinhardtii-PSII WOC cycles less efficiently at all light intensities and produces less O(2) than either cyanobacterial D1 isoform. D1:2-PSII makes more O(2) per saturating flash than D1:1-PSII, but it exhibits lower WOC cycling efficiency at low light intensities due to a 40% faster charge recombination rate in the S(3) state. These functional advantages of D1:1-PSII and D1:2-PSII at low and high light regimes, respectively, can be explained by differences in predicted redox potentials of PSII electron acceptors that control kinetic performance.
Vondenhoff, GHM, Dubiley S, Severinov K, Lescrinier E, Rozenski J, Van Aerschot A.  2011.  Extended targeting potential and improved synthesis of Microcin C analogues as antibacterials. Bioorg. & Med. Chem.. 19:5462-5467.
Vondenhoff, GHM, Blanchaert B, Geboers S, Kazakov TS, Severinov K, Van Aerschot A.  2011.  Synthesis and evaluation of Microcin C analogues containing various peptide chains. J. Bacteriol.. 193:3618-3623.
Vorobiev, SM, Gensler Y, Vahedian-Movahed H, Seetharaman J, Su M, Huang JY, Xiao R, Kornhaber G, Montelione GT, Tong L et al..  2014.  Structure of the DNA-Binding and RNA-Polymerase-Binding Region of Transcription Antitermination Factor λQ.. Structure . 22:485-495. Abstract
The bacteriophage λ Q protein is a transcription antitermination factor that controls expression of the phage late genes as a stable component of the transcription elongation complex. To join the elongation complex, λQ binds a specific DNA sequence element and interacts with RNA polymerase that is paused during early elongation. λQ binds to the paused early-elongation complex through interactions between λQ and two regions of RNA polymerase: region 4 of the σ(70) subunit and the flap region of the β subunit. We present the 2.1 Å resolution crystal structure of a portion of λQ containing determinants for interaction with DNA, interaction with region 4 of σ(70), and interaction with the β flap. The structure provides a framework for interpreting prior genetic and biochemical analysis and sets the stage for future structural studies to elucidate the mechanism by which λQ alters the functional properties of the transcription elongation complex.
Vorobiev, SM, Gensler Y, Vahedian-Movahed H, Seetharaman J, Su M, Huang JY, Xiao R, Kornhaber G, Montelione GT, Tong L et al..  2014.  Structure of the DNA-binding and RNA polymerase-binding region of transcription antitermination factor λQ. Structure. 22:488-495.
Vrentas, CE, Gaal T, Ross W, Ebright RH, Gourse RL.  2005.  Response of RNA polymerase to ppGpp: requirement for the omega subunit and relief of this requirement by DksA.. Genes & development. 19(19):2378-87. Abstract
Previous studies have come to conflicting conclusions about the requirement for the omega subunit of RNA polymerase in bacterial transcription regulation. We demonstrate here that purified RNAP lacking omega does not respond in vitro to the effector of the stringent response, ppGpp. DksA, a transcription factor that works in concert with ppGpp to regulate rRNA expression in vivo and in vitro, fully rescues the ppGpp-unresponsiveness of RNAP lacking omega, likely explaining why strains lacking omega display a stringent response in vivo. These results demonstrate that omega plays a role in RNAP function (in addition to its previously reported role in RNAP assembly) and highlight the importance of inclusion of omega in RNAP purification protocols. Furthermore, these results suggest that either one or both of two short segments in the beta' subunit that physically link omega to the ppGpp-binding region of the enzyme may play crucial roles in ppGpp and DksA function.
Vvedenskaya, IO, Vahedian-Movahed H, Bird JG, Knoblauch JG, Goldman SR, Zhang Y, Ebright RH, Nickels BE.  2014.  Interactions between RNA polymerase and the "core recognition element" counteract pausing. Science. 344(6189):1285-1289. Abstract
Transcription elongation is interrupted by sequences that inhibit nucleotide addition and cause RNA polymerase (RNAP) to pause. Here, by use of native elongating transcript sequencing (NET-seq) and a variant of NET-seq that enables analysis of mutant RNAP derivatives in merodiploid cells (mNET-seq), we analyze transcriptional pausing genome-wide in vivo in Escherichia coli. We identify a consensus pause-inducing sequence element, G₋₁₀Y₋₁G(+1) (where -1 corresponds to the position of the RNA 3' end). We demonstrate that sequence-specific interactions between RNAP core enzyme and a core recognition element (CRE) that stabilize transcription initiation complexes also occur in transcription elongation complexes and facilitate pause read-through by stabilizing RNAP in a posttranslocated register. Our findings identify key sequence determinants of transcriptional pausing and establish that RNAP-CRE interactions modulate pausing.
Vvedenskaya, IO, Vahedian-Movahed H, Bird JG, Knoblauch JG, Goldman SR, Zhang Y, Ebright RH, Nickels BE.  2014.  Transcription. Interactions between RNA polymerase and the "core recognition element" counteract pausing.. Science (New York, N.Y.). 344(6189):1285-9. AbstractWebsite
Transcription elongation is interrupted by sequences that inhibit nucleotide addition and cause RNA polymerase (RNAP) to pause. Here, by use of native elongating transcript sequencing (NET-seq) and a variant of NET-seq that enables analysis of mutant RNAP derivatives in merodiploid cells (mNET-seq), we analyze transcriptional pausing genome-wide in vivo in Escherichia coli. We identify a consensus pause-inducing sequence element, G₋₁₀Y₋₁G(+1) (where -1 corresponds to the position of the RNA 3' end). We demonstrate that sequence-specific interactions between RNAP core enzyme and a core recognition element (CRE) that stabilize transcription initiation complexes also occur in transcription elongation complexes and facilitate pause read-through by stabilizing RNAP in a posttranslocated register. Our findings identify key sequence determinants of transcriptional pausing and establish that RNAP-CRE interactions modulate pausing.
Vvedenskaya, IO, Sharp JS, Goldman SR, Kanabar PN, Livny J, Dove SL, Nickels BE.  2012.  Growth phase-dependent control of transcription start site selection and gene expression by nanoRNAs. Genes & development. 26:1498-507. AbstractWebsite
Prokaryotic and eukaryotic RNA polymerases can use 2- to approximately 4-nt RNAs, "nanoRNAs," to prime transcription initiation in vitro. It has been proposed that nanoRNA-mediated priming of transcription can likewise occur under physiological conditions in vivo and influence transcription start site selection and gene expression. However, no direct evidence of such regulation has been presented. Here we demonstrate in Escherichia coli that nanoRNAs prime transcription in a growth phase-dependent manner, resulting in alterations in transcription start site selection and changes in gene expression. We further define a sequence element that determines, in part, whether a promoter will be targeted by nanoRNA-mediated priming. By establishing that a significant fraction of transcription initiation is primed in living cells, our findings contradict the conventional model that all cellular transcription is initiated using nucleoside triphosphates (NTPs) only. In addition, our findings identify nanoRNAs as a previously undocumented class of regulatory small RNAs that function by being directly incorporated into a target transcript.
Vvedenskaya, IO, Goldman SR, Nickels BE.  2015.  Preparation of cDNA libraries for high-throughput RNA sequencing analysis of RNA 5' ends. Methods Mol Biol. 1276:211-228.
Vvedenskaya, IO, Sharp JS, Goldman SR, Kanabar PN, Livny J, Dove SL, Nickels BE.  2012.  Growth phase-dependent control of transcription start site selection and gene expression by nanoRNAs. Genes Dev. 26(13):1498-1507. Abstract
Prokaryotic and eukaryotic RNA polymerases can use 2- to ~4-nt RNAs, ‘‘nanoRNAs,’’ to prime transcription initiation in vitro. It has been proposed that nanoRNA-mediated priming of transcription can likewise occur under physiological conditions in vivo and influence transcription start site selection and gene expression. However, no direct evidence of such regulation has been presented. Here we demonstrate in Escherichia coli that nanoRNAs prime transcription in a growth phase-dependent manner, resulting in alterations in transcription start site selection and changes in gene expression. We further define a sequence element that determines, in part, whether a promoter will be targeted by nanoRNA-mediated priming. By establishing that a significant fraction of transcription initiation is primed in living cells, our findings contradict the conventional model that all cellular transcription is initiated using nucleoside triphosphates (NTPs) only. In addition, our findings identify nanoRNAs as a previously undocumented class of regulatory small RNAs that function by being directly incorporated into a target transcript.
Vvedenskaya, IO, Vahedian-Movahed H, Zhang Y, Taylor DM, Ebright RH, Nickels BE.  2016.  Interactions between RNA polymerase and the core recognition element are a determinant of transcription start site selection.. Proc. Natl. Acad. Sci. U.S.A. 113(21):E2899-2905.
Vvedenskaya, IO, Zhang Y, Goldman SR, Valenti A, Visone V, Taylor DM, Ebright RH, Nickels BE.  2015.  Massively Systematic Transcript End Readout, "MASTER": Transcription Start Site Selection, Transcriptional Slippage, and Transcript Yields.. Molecular cell. 60:953-965. Abstract
We report the development of a next-generation sequencing-based technology that entails construction of a DNA library comprising up to at least 4(7) (∼16,000) barcoded sequences, production of RNA transcripts, and analysis of transcript ends and transcript yields (massively systematic transcript end readout, "MASTER"). Using MASTER, we define full inventories of transcription start sites ("TSSomes") of Escherichia coli RNA polymerase for initiation at a consensus core promoter in vitro and in vivo; we define the TSS-region DNA sequence determinants for TSS selection, reiterative initiation ("slippage synthesis"), and transcript yield; and we define effects of DNA topology and NTP concentration. The results reveal that slippage synthesis occurs from the majority of TSS-region DNA sequences and that TSS-region DNA sequences have profound, up to 100-fold, effects on transcript yield. The results further reveal that TSSomes depend on DNA topology, consistent with the proposal that TSS selection involves transcription-bubble expansion ("scrunching") and transcription-bubble contraction ("anti-scrunching").
Vvedenskaya, IO, Vahedian-Movahed H, Zhang Y, Taylor DM, Ebright RH, Nickels BE.  2016.  Interactions between RNA polymerase and the core recognition element are a determinant of transcription start site selection.. Proceedings of the National Academy of Sciences of the United States of America. 113:E2899-E2905. Abstract
During transcription initiation, RNA polymerase (RNAP) holoenzyme unwinds ∼13 bp of promoter DNA, forming an RNAP-promoter open complex (RPo) containing a single-stranded transcription bubble, and selects a template-strand nucleotide to serve as the transcription start site (TSS). In RPo, RNAP core enzyme makes sequence-specific protein-DNA interactions with the downstream part of the nontemplate strand of the transcription bubble ("core recognition element," CRE). Here, we investigated whether sequence-specific RNAP-CRE interactions affect TSS selection. To do this, we used two next-generation sequencing-based approaches to compare the TSS profile of WT RNAP to that of an RNAP derivative defective in sequence-specific RNAP-CRE interactions. First, using massively systematic transcript end readout, MASTER, we assessed effects of RNAP-CRE interactions on TSS selection in vitro and in vivo for a library of 4(7) (∼16,000) consensus promoters containing different TSS region sequences, and we observed that the TSS profile of the RNAP derivative defective in RNAP-CRE interactions differed from that of WT RNAP, in a manner that correlated with the presence of consensus CRE sequences in the TSS region. Second, using 5' merodiploid native-elongating-transcript sequencing, 5' mNET-seq, we assessed effects of RNAP-CRE interactions at natural promoters in Escherichia coli, and we identified 39 promoters at which RNAP-CRE interactions determine TSS selection. Our findings establish RNAP-CRE interactions are a functional determinant of TSS selection. We propose that RNAP-CRE interactions modulate the position of the downstream end of the transcription bubble in RPo, and thereby modulate TSS selection, which involves transcription bubble expansion or transcription bubble contraction (scrunching or antiscrunching).
Vvedenskaya, IO, Zhang Y, Goldman SR, Valenti A, Visone V, Taylor DM, Ebright RH, Nickels BE.  2015.  Massively systematic transcript end readout, “MASTER”: transcription start site selection, transcriptional slippage, and transcript yields. Molecular Cell. 60:953-965.