The nucleotide addition cycle of RNA polymerase is controlled by two molecular hinges in the Bridge Helix domain

Abstract Background Cellular RNA polymerases (RNAPs) are complex molecular machines that combine catalysis with concerted conformational changes in the active center. Previous work showed that kinking of a hinge region near the C-terminus of the Bridge Helix (BH-HC) plays a critical role in controlling the catalytic rate. Results Here, new evidence for the existence of an additional hinge region in the amino-terminal portion of the Bridge Helix domain (BH-HN) is presented. The nanomechanical properties of BH-HN emerge as a direct consequence of the highly conserved primary amino acid sequence. Mutations that are predicted to influence its flexibility cause corresponding changes in the rate of the nucleotide addition cycle (NAC). BH-HN displays functional properties that are distinct from BH-HC, suggesting that conformational changes in the Bridge Helix control the NAC via two independent mechanisms. Conclusions The properties of two distinct molecular hinges in the Bridge Helix of RNAP determine the functional contribution of this domain to key stages of the NAC by coordinating conformational changes in surrounding domains.</p

TraR, a Homolog of a RNAP Secondary Channel Interactor, Modulates Transcription

Recent structural and biochemical studies have identified a novel control mechanism of gene expression mediated through the secondary channel of RNA Polymerase (RNAP) during transcription initiation. Specifically, the small nucleotide ppGpp, along with DksA, a RNAP secondary channel interacting factor, modifies the kinetics of transcription initiation, resulting in, among other events, down-regulation of ribosomal RNA synthesis and up-regulation of several amino acid biosynthetic and transport genes during nutritional stress. Until now, this mode of regulation of RNAP was primarily associated with ppGpp. Here, we identify TraR, a DksA homolog that mimics ppGpp/DksA effects on RNAP. First, expression of TraR compensates for dksA transcriptional repression and activation activities in vivo. Second, mutagenesis of a conserved amino acid of TraR known to be critical for DksA function abolishes its activity, implying both structural and functional similarity to DksA. Third, unlike DksA, TraR does not require ppGpp for repression of the rrnB P1 promoter in vivo and in vitro or activation of amino acid biosynthesis/transport genes in vivo. Implications for DksA/ppGpp mechanism and roles of TraR in horizontal gene transfer and virulence are discussed

EPHA2 Is Associated with Age-Related Cortical Cataract in Mice and Humans

Age-related cataract is a major cause of blindness worldwide, and cortical cataract is the second most prevalent type of age-related cataract. Although a significant fraction of age-related cataract is heritable, the genetic basis remains to be elucidated. We report that homozygous deletion of Epha2 in two independent strains of mice developed progressive cortical cataract. Retroillumination revealed development of cortical vacuoles at one month of age; visible cataract appeared around three months, which progressed to mature cataract by six months. EPHA2 protein expression in the lens is spatially and temporally regulated. It is low in anterior epithelial cells, upregulated as the cells enter differentiation at the equator, strongly expressed in the cortical fiber cells, but absent in the nuclei. Deletion of Epha2 caused a significant increase in the expression of HSP25 (murine homologue of human HSP27) before the onset of cataract. The overexpressed HSP25 was in an underphosphorylated form, indicating excessive cellular stress and protein misfolding. The orthologous human EPHA2 gene on chromosome 1p36 was tested in three independent worldwide Caucasian populations for allelic association with cortical cataract. Common variants in EPHA2 were found that showed significant association with cortical cataract, and rs6678616 was the most significant in meta-analyses. In addition, we sequenced exons of EPHA2 in linked families and identified a new missense mutation, Arg721Gln, in the protein kinase domain that significantly alters EPHA2 functions in cellular and biochemical assays. Thus, converging evidence from humans and mice suggests that EPHA2 is important in maintaining lens clarity with age

Transcriptional control in the prereplicative phase of T4 development

Control of transcription is crucial for correct gene expression and orderly development. For many years, bacteriophage T4 has provided a simple model system to investigate mechanisms that regulate this process. Development of T4 requires the transcription of early, middle and late RNAs. Because T4 does not encode its own RNA polymerase, it must redirect the polymerase of its host, E. coli, to the correct class of genes at the correct time. T4 accomplishes this through the action of phage-encoded factors. Here I review recent studies investigating the transcription of T4 prereplicative genes, which are expressed as early and middle transcripts. Early RNAs are generated immediately after infection from T4 promoters that contain excellent recognition sequences for host polymerase. Consequently, the early promoters compete extremely well with host promoters for the available polymerase. T4 early promoter activity is further enhanced by the action of the T4 Alt protein, a component of the phage head that is injected into E. coli along with the phage DNA. Alt modifies Arg265 on one of the two α subunits of RNA polymerase. Although work with host promoters predicts that this modification should decrease promoter activity, transcription from some T4 early promoters increases when RNA polymerase is modified by Alt. Transcription of T4 middle genes begins about 1 minute after infection and proceeds by two pathways: 1) extension of early transcripts into downstream middle genes and 2) activation of T4 middle promoters through a process called sigma appropriation. In this activation, the T4 co-activator AsiA binds to Region 4 of σ70, the specificity subunit of RNA polymerase. This binding dramatically remodels this portion of σ70, which then allows the T4 activator MotA to also interact with σ70. In addition, AsiA restructuring of σ70 prevents Region 4 from forming its normal contacts with the -35 region of promoter DNA, which in turn allows MotA to interact with its DNA binding site, a MotA box, centered at the -30 region of middle promoter DNA. T4 sigma appropriation reveals how a specific domain within RNA polymerase can be remolded and then exploited to alter promoter specificity

Relação entre coeficientes de cultura e graus-dia de desenvolvimento da alface Relationship between lettuce crop coeficient and growing degree days

Este estudo foi conduzido na Fazenda Campbell da Universidade do Arizona , no período de junho de 1994 a fevereiro de 1995, com objetivo de determinar a equação do coeficiente de cultura (Kc) como função de graus-dia de desenvolvimento (GDD) usando a série de senos Fourier, para alface (Lactuca sativa, L.), do tipo folhosa. Foram usadas dez parcelas casualizadas, irrigadas de maneira a manter o potencial matricial da água no solo acima ou igual a -20kPa, que constituiram as repetições na determinação dos valores de evapotranspiração máxima da cultura (ETm). Cada parcela foi constituida por seis canteiros de 1,0 m x 7,3 m, com duas linhas de plantas (cultivar Waldmann's Green) em cada, com população, após desbaste, de 48 plantas por canteiro. A época de desbaste ocorreu com GDD de 228ºC e Kc médio de 0,4 e a colheita com GDD igual a 742ºC e Kc médio de 1,2. Os valores de Kc como função de GDD, em diferentes estádios do ciclo da cultura, permitiram gerar uma equação que pode ser inserida em programa de computador para manejo de irrigação, considerando um GDD final (GDD de ajuste) de 900ºC.<br>This research was carried out at the Campbell Farm of The University of Arizona, from June 1994 to February 1995, with the aim of determining a growing-degree-days-based crop coefficient (Kc) equation for leaf lettuce (Lactuca sativa L.), using the Fourier sine series model. Ten randomized experimental plots were irrigated, in order to maintain a soil matric potential greater or equal to -20 kPa, as replications for the determination of crop maximum evapotranspiration (ETm). Each plot consisted of six raised beds, 7.3 m long and 1.0 m wide, with two rows of lettuce (Lactuca sativa L., cv. Waldmann's Green) thinned to a final population of 48 plants per bed. The thinning time occurred with a GDD of 228ºC and average Kc of 0.4. The harvest time occurred with GDD of 742ºC and average Kc of 1.2. The values of Kc as function of GDD, during the crop season, allowed the generation of an equation that can be inserted into irrigation scheduling software, considering an ending GDD (adjustment GDD) of 900ºC

Impact of template backbone heterogeneity on RNA polymerase II transcription

Variations in the sugar component (ribose or deoxyri-bose) and the nature of the phosphodiester linkage (3′-5 ′ or 2′-5 ′ orientation) have been a challenge for genetic information transfer from the very beginning of evolution. RNA polymerase II (pol II) governs the transcription of DNA into precursor mRNA in all eu-karyotic cells. How pol II recognizes DNA template backbone (phosphodiester linkage and sugar) and whether it tolerates the backbone heterogeneity re-main elusive. Such knowledge is not only important for elucidating the chemical basis of transcriptional fidelity but also provides new insights into molecular evolution. In this study, we systematically and quan-titatively investigated pol II transcriptional behaviors through different template backbone variants. We re-vealed that pol II can well tolerate and bypass sugar heterogeneity sites at the template but stalls at phos-phodiester linkage heterogeneity sites. The distinct impacts of these two backbone components on pol II transcription reveal the molecular basis of template recognition during pol II transcription and provide the evolutionary insight from the RNA world to the contemporary ‘imperfect ’ DNA world. In addition, our results also reveal the transcriptional consequences from ribose-containing genomic DNA