Structure of the DNA-binding and RNA-polymerase-binding region of transcription antitermination factor λQ.

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 ß 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 ß 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.

DNA methylation, riboswitches, and transcription factor activity: fundamental mechanisms of gene-nutrient interactions involving vitamins.

Nutrient-gene interactions occur with a variety of nutrients including some minerals, vitamins, polyunsaturated fatty acids and other lipids. Fundamental molecular mechanisms that underlie many of the effects of nutrients on gene expression are presented herein. Two of the mechanisms described influence gene transcription: DNA methylation and transcription factor activation. Another mechanism, riboswitching, can regulate gene expression at different levels, for example, at the mRNA translation level. The first two mechanisms are widely distributed across animal phyla. Riboswitches are documented primarily in more primitive organisms, but may prove to be of wider relevance. Riboswitches are known for several vitamins; those involving thiamine are presented here. The role of folates and retinoids in DNA methylation and transcriptional factor (nuclear retinoid receptor) activities, respectively, is presented in the context of cell proliferation and differentiation, and related physiological or pathological effects during embryogenesis and cancer.

Evidence for a specific cell membrane retinol-binding protein transport mechanism in a human keratinocyte line.

The epidermis is highly sensitive to retinoids, and vitamin A (retinol) is a critical factor in the regulation of skin cell differentiation and proliferation. Despite extensive knowledge of retinoid-mediated gene transcription effects on epidermal cells and evidence for retinoid-mediated suppression of carcinogenesis in skin, basic transport events, especially cellular uptake, of this vitamin remain poorly understood and controversial. Herein, evidence is presented for receptor-mediated uptake of retinol-binding protein, RBP, the specific circulatory vitamin A carrier, in the A431 human epidermal cell line. Cellular RBP uptake was significantly inhibited by anti-RBP IgG. Addition of transthyretin (TTR), a circulatory protein that can interact with RBP, to the internalization assay also significantly reduced RBP uptake to 49.4+/-4.6% (+/- SEM) of control values (p<0.01). RBP uptake was impaired by sucrose, a known inhibitor of early endocytosis, but not significantly affected by a disruptor of later trafficking events, chlorpromazine. Binding analysis indicated saturable RBP binding to the cell surface and a total of about 94,000 binding sites/cell. Based on dissociation constants, two RBP binding sites were detected with a 50-fold affinity difference: 0.7 and 35.0 nM, with 12,000 and 82,000 receptors/cell, respectively. These results indicate that high affinity RBP receptors capable of internalizing RBP independently of TTR exist in these malignant keratinocytes, and that TTR influences binding of RBP to its putative receptor(s). Overall, the data establish membrane transport parameters for RBP, and provide a basis for examining modulation of vitamin A endocytosis that may accompany changes in proliferation or differentiation state of epidermal cells.

The clustering of neonatal deaths in triplet pregnancies: application of response conditional multivariate logistic regression models.

BACKGROUND/OBJECTIVES: A population-based retrospective cohort study of triplet pregnancies was conducted to estimate individual probabilities of neonatal mortality (death within 28 days of birth) conditional on the number of neonatal deaths experienced by other infants in the triplet set. METHODS: Data on 4,697 triplet sets (14,091 births) were derived from the U.S. 1995-1997 matched multiple birth file assembled by the National Center for Health Statistics. Response conditional multivariate logistic regression was used to model the association of neonatal mortality among cotriplets. To account for the correlation of the outcomes among cotriplets, regression parameters were estimated by the methodology of generalized estimating equations with robust variance estimates. RESULTS: Compared with a triplet where both cotriplets survived the neonatal period, the adjusted odds ratio and 95% confidence interval (CI) for a neonatal death associated with one and two cotriplet neonatal deaths were 1.80 (95% CI 1.06, 3.04), and 13.41 (95% CI 2.31, 77.7), respectively, after adjusting for birthweight and gestational age. CONCLUSIONS: These results show strong evidence of clustering of neonatal deaths in triplet pregnancies.

Structure of antibacterial peptide microcin J25: a 21-residue lariat protoknot.

The antibacterial peptide microcin J25 (MccJ25) inhibits bacterial transcription by binding within, and obstructing, the nucleotide-uptake channel of bacterial RNA polymerase. Published covalent and three-dimensional structures indicate that MccJ25 is a 21-residue cycle. Here, we show that the published covalent and three-dimensional structures are incorrect, and that MccJ25 in fact is a 21-residue "lariat protoknot", consisting of an 8-residue cyclic segment followed by a 13-residue linear segment that loops back and threads through the cyclic segment. MccJ25 is the first example of a lariat protoknot involving a backbone-side chain amide linkage.