The TATA-binding protein (TBP) initiates assembly of transcription preinitiation complexes on eukaryotic class II promoters, binding to and restructuring consensus and variant "TATA box" sequences. The sequence dependence of the DNA structure in TBP⅐TATA complexes has been investigated in solution using fluorescence resonance energy transfer. The mean 5dye-3dye distance varies significantly among oligomers bearing the adenovirus major late promoter sequence (AdMLP) and five single-site variants bound to Saccharomyces cerevisiae TBP, consistent with solution bend angles for AdMLP of 76°and for the variants ranging from 30°to 62°. These solution bends contrast sharply with the corresponding co-crystal structures, which show ϳ80°b ends for all sequences. Transcription activities for these TATA sequences are strongly correlated with the solution bend angles but not with TBP⅐DNA binding affinities. Our results support a model in which transcription efficiency derives primarily from the sequence-dependent structure of the TBP⅐TATA binary complex. Specifically, the distance distribution for the average solution structure of the TBP⅐TATA complex may reflect the sequence-dependent probability for the complex to assume a conformation in which the TATA box DNA is severely bent. Upon assumption of this geometry, the binary complex becomes a target for binding and correctly orienting the other components of the preinitiation complex.The TATA-binding protein (TBP) 1 binds to eukaryotic class II promoters at specific sequences of DNA of the consensus sequence TATA(a/t)A(a/t)N, nucleating assembly of the proteins required for transcription. Atomic resolution co-crystal structures of complexes of DNA bearing consensus strong promoter sequences bound to Saccharomyces cerevisiae (1), Arabidopsis thaliana (2), and human (3, 4) TBPs are extremely similar, characterized by a TBP-induced ϳ80°bend in the DNA helix. TBP also binds to numerous variant TATA sequences, many of which occur naturally in promoters (5, 6). For 21 such single-point mutants of the adenovirus major late promoter (AdMLP) TATA box sequence, in vitro transcription activity was found to range from Ͻ1% to 107% of that of the reference AdMLP TATA sequence (6).The wide range of observed transcription activities suggested that TBP does not bind similarly to all TATA elements. Gel electrophoresis circular permutation analysis of TBP⅐DNA complexes shows that the electrophoretic mobility of the complexes is TATA sequence-dependent, with bend angles from Ͻ34°to 106°inferred from the gel mobility patterns (7). In contrast, the co-crystal structures of 11 TATA sequence variants of varying affinity bound to A. thaliana TBP are all very similar, with the DNA helix bent as in the strong promoters (3,8).The present study was undertaken to further explore the TATA box sequence dependence of TBP binding and DNA structure using native, full-length S. cerevisiae TBP together with the AdMLP TATA sequence and five single-base-pair variant sequences. End-to-end distance distributions for...

SummaryBacteria of the SAR11 clade constitute up to one half of all microbial cells in the oxygen-rich surface ocean. DNA sequences from SAR11 are also abundant in oxygen minimum zones (OMZs) where oxygen falls below detection and anaerobic microbes play important roles in converting bioavailable nitrogen to N2 gas. Evidence for anaerobic metabolism in SAR11 has not yet been observed, and the question of how these bacteria contribute to OMZ biogeochemical cycling is unanswered. Here, we identify the metabolic basis for SAR11 activity in anoxic ocean waters. Genomic analysis of single cells from the world’s largest OMZ revealed diverse and previously uncharacterized SAR11 lineages that peak in abundance at anoxic depths, but are largely undetectable in oxygen-rich ocean regions. OMZ SAR11 contain adaptations to low oxygen, including genes for respiratory nitrate reductases (Nar). SAR11 nar genes were experimentally verified to encode proteins catalyzing the nitrite-producing first step of denitrification and constituted ~40% of all OMZ nar transcripts, with transcription peaking in the zone of maximum nitrate reduction rates. These results redefine the ecological niche of Earth’s most abundant organismal group and suggest an important contribution of SAR11 to nitrite production in OMZs, and thus to pathways of ocean nitrogen loss.

Alterations in homocysteine, methionine, folate, and/or B 12 homeostasis have been associated with neural tube defects, cardiovascular disease, and cancer. Methionine synthase, one of only two mammalian enzymes known to require vitamin B 12 as a cofactor, lies at the intersection of these metabolic pathways. This enzyme catalyzes the transfer of a methyl group from 5-methyl-tetrahydrofolate to homocysteine, generating tetrahydrofolate and methionine. Human patients with methionine synthase deficiency exhibit homocysteinemia, homocysteinuria, and hypomethioninemia. They suffer from megaloblastic anemia with or without some degree of neural dysfunction and mental retardation. To better study the pathophysiology of methionine synthase deficiency, we utilized gene-targeting technology to inactivate the methionine synthase gene in mice. On average, heterozygous knockout mice from an outbred background have slightly elevated plasma homocysteine and methionine compared to wild-type mice but seem to be otherwise indistinguishable. Homozygous knockout embryos survive through implantation but die soon thereafter. Nutritional supplementation during pregnancy was unable to rescue embryos that were completely deficient in methionine synthase. Whether any human patients with methionine synthase deficiency have a complete absence of enzyme activity is unclear. These results demonstrate the importance of this enzyme for early development in mice and suggest either that methionine synthasedeficient patients have residual methionine synthase activity or that humans have a compensatory mechanism that is absent in mice.Methionine synthase (MS; EC 2.1.1.13), one of two B 12 -dependent mammalian enzymes, catalyzes the remethylation of homocysteine to methionine and the concurrent demethylation of 5-methyltetrahydrofolate (5-Me-THF) to tetrahydrofolate (THF). Methylcobalamin, a derivative of vitamin B 12 , is the cofactor for this reaction. MSs are highly conserved, large (about 140-kDa) monomeric Zn metalloproteins and consist of three domains: a catalytic domain that contains the binding sites for 5-Me-THF and homocysteine; a B 12 domain, where the methylcobalamin cofactor is tightly bound; and an activation domain. The cob(I)alamin form of the cofactor is methylated by 5-Me-THF, generating enzyme-bound methylcob (III)alamin and THF. Then methylcob(III)alamin transfers its methyl group to homocysteine to produce methionine and returns to the cob(I)alamin form. Occasionally, the highly reactive cob(I)alamin cofactor is oxidized to the nonfunctional cob(II)alamin form during catalysis. The enzyme is reactivated by an S-adenosylmethionine (AdoMet) and NADPH-dependent reductive methylation of enzyme-bound cob(II)alamin to methylcob(III)alamin. The enzyme(s) involved in this reductive methylation has not been well characterized, and two pathways have been proposed. The first pathway involves a single protein, MS reductase, a soluable P 450 -reductase-like protein that contains binding sites for NADPH, flavin adenine dinucleotide, and fla...

The complex formed between the TATA-binding protein (TBP) and the "TATA box" of eukaryotic class II promoters is the foundation for assembly of the complex to which RNA polymerase II is ultimately recruited. TBP binds productively to canonical and diverse variant TATA sequences with >100-fold differences in transcription efficiency. Co-crystals of canonical sequences and >11 variant sequences bound to various TBP molecules all have ϳ80°bends. In contrast, the bend angles for TBP⅐TATA complexes in solution, derived from distance distributions, are ϳ80°for a canonical sequence but range from 30°to 62°for five variant sequences (1). We show in this study that the osmolytes used to crystallize TBP⅐TATA complexes induce profound increases in the DNA bends of two transcriptionally active TBP-bound variant sequences to a common angle of ϳ80°but have little effect on a transcriptionally inactive variant. The effect of osmolyte on the TBP-induced DNA bend of a variant TATA box sequence is also manifest in the kinetics of association, demonstrating a functional consequence of an osmolyte-induced structural change.The complex formed between the TATA-binding protein (TBP) 1 and eukaryotic class II promoters of consensus sequence TATA(a/t)A(a/t)N is the foundation for assembly of the preinitiation complex to which RNA polymerase II is ultimately recruited. TBP binds productively to canonical and diverse variant TATA sequences with Ͼ100-fold differences in transcription efficiency (2). A key feature of the solution geometry of the adenovirus major late promoter (AdMLP) and five variant sequences bound to Saccharomyces cerevisiae TBP has been determined using time-resolved fluorometry coupled with För-ster resonance energy transfer (FRET) (1). The mean end-toend distances in these six TBP-bound sequences vary widely and correspond to TBP-induced bends of 76°for the consensus AdMLP sequence and from 30°to 62°for the variant sequences.High resolution co-crystal structures of canonical TATA sequences bound to S. cerevisiae (3), Arabidopsis thaliana (4), and human (5, 6) TBPs are extremely similar. These binary structures are characterized by ϳ80°-induced bends in the DNA helical axes, in excellent accord with the solution bend angle obtained subsequently for AdMLP using FRET. However, in contrast to the bends determined using FRET, the co-crystal structures of 11 TATA sequence variants (including those examined by FRET) bound to A. thaliana TBP are all very similar, with the DNA bent ϳ80°as in the strong promoters (6, 7).Because assembly of the transcription complex proceeds by sequential structural alterations, a clear picture of the relevant discrete structures is fundamental to an accurate understanding of these processes. The present study was therefore undertaken to further investigate the relationship between the solution and co-crystal structures of TBP⅐promoter complexes.Osmolytes used as crystallizing agents for TBP⅐TATA complexes were postulated to induce in the bound variant sequences severe bends approaching those o...

SUMMARYA method for direct numerical analysis of three-dimensional deformable particles suspended in fluid is presented. The flow is computed on a fixed regular 'lattice' using the lattice Boltzmann method (LBM), where each solid particle is mapped onto a Lagrangian frame moving continuously through the domain. Instead of the bounce-back method, an external boundary force (EBF) is used to impose the no-slip boundary condition at the fluid-solid interface for stationary or moving boundaries. The EBF is added directly to the lattice Boltzmann equation. The motion and orientation of the particles are obtained from Newtonian dynamics equations. The advantage of this approach is outlined in comparison with the standard and higher-order interpolated bounce-back methods as well as the LBM immersed-boundary and the volume-of-fluid methods. Although the EBF method is general, in this application, it is used in conjunction with the lattice-spring model for deformable particles. The methodology is validated by comparing with experimental and theoretical results.