Mapping the active site of yeast RNA polymerase B (II)

Yeast RNA polymerase B (II) was incubated with a collection of 13 different nucleotide derivatives and affinity labeled by allowing DNA-directed phosphodiester bond formation. The 32P-labeled site was localized in the C-terminal part of the B150 subunit by microsequencing a proteolytic fragment, then further mapped by a combination of extensive or single-hit chemical cleavage reactions and analysis of the labeled peptide patterns. The affinity label was mapped to between Asn946 and Met999, within one of the nine regions that are conserved between B150 and the bacterial beta subunit. The results underscore the conservative evolution of the catalytic center of eukaryotic and bacterial RNA polymerases.     

The lymphocyte receptor CD6 interacts with syntenin-1, a scaffolding protein containing PDZ domains

CD6 is a type I membrane glycoprotein expressed on thymocytes, mature T and B1a lymphocytes, and CNS cells. CD6 binds to activated leukocyte cell adhesion molecule (CD166), and is considered as a costimulatory molecule involved in lymphocyte activation and thymocyte development. Accordingly, CD6 partially associates with the TCR/CD3 complex and colocalizes with it at the center of the mature immunological synapse (IS) on T lymphocytes. However, the signaling pathway used by CD6 is still mostly unknown. The yeast two-hybrid system has allowed us the identification of syntenin-1 as an interacting protein with the cytoplasmic tail of CD6. Syntenin-1 is a PDZ (postsynaptic density protein-95, postsynaptic discs large, and zona occludens-1) domain-containing protein, which functions as an adaptor protein able to bind cytoskeletal proteins and signal transduction effectors. Mutational analyses showed that certain amino acids of the most C-terminal sequence of CD6 (-YDDISAA) and the two postsynaptic density protein-95, postsynaptic discs large, and zona occludens-1 domains of syntenin-1 are relevant to the interaction. Further confirmation of the CD6-syntenin-1 interaction was obtained from pull-down and co-immunoprecipitation assays in mammalian cells. Image analyses also showed that syntenin-1 accumulates at CD6 caps and at the IS. Therefore, we propose that syntenin-1 may function as a scaffolding protein coupling CD6 and most likely other lymphocyte receptors to cytoskeleton and/or signaling effectors during IS maturation.     

A Monoclonal Antibody that Specifically Recognizes m'A Nucleoside

Abstract

A hybridoma against the nucleoside m⁶A has been obtained from mouse spleen. This hybridoma was named H65 and it secretes monoclonal antibodies anti-m⁶A. The competition assays showed that the monoclonal antibody was highly specific for m⁶A nucleoside.     

Structure-Function Features of a Mycoplasma Glycolipid Synthase Derived from Structural Data Integration,Molecular Simulations,and Mutational Analysis

Glycoglycerolipids are structural components of mycoplasma membranes with a fundamental role in membrane properties and stability. Their biosynthesis is mediated by glycosyltransferases (GT) that catalyze the transfer of glycosyl units from a sugar nucleotide donor to diacylglycerol. The essential function of glycolipid synthases in mycoplasma viability, and the absence of glycoglycerolipids in animal host cells make these GT enzymes a target for drug discovery by designing specific inhibitors. However, rational drug design has been hampered by the lack of structural information for any mycoplasma GT. Most of the annotated GTs in pathogenic mycoplasmas belong to family GT2. We had previously shown that MG517 in Mycoplasma genitalium is a GT-A family GT2 membrane-associated glycolipid synthase. We present here a series of structural models of MG517 obtained by homology modeling following a multiple-template approach. The models have been validated by mutational analysis and refined by long scale molecular dynamics simulations. Based on the models, key structure-function relationships have been identified: The N-terminal GT domain has a GT-A topology that includes a non-conserved variable region involved in acceptor substrate binding. Glu193 is proposed as the catalytic base in the GT mechanism, and Asp40, Tyr126, Tyr169, Ile170 and Tyr218 define the substrates binding site. Mutation Y169F increases the enzyme activity and significantly alters the processivity (or sequential transferase activity) of the enzyme. This is the first structural model of a GT-A glycoglycerolipid synthase and provides preliminary insights into structure and function relationships in this family of enzymes.     

Evolutionary patterns of Clavatoraceae (Charophyta) in the mesogean basins analysed according to environmental change during Malm and lower cretaceous

Evolutionary patterns of Clavatoraceae during the Malm and the Lower Cretaceous can be understood by considering how palaeoecological constraints of these charophytes were affected by palaeoenvironmental change. Speciation of Clavatoraceae reached maxima in the Tithonian and especially in the Lower Barremian, coinciding with an important areal extension and environmental diversification of freshwater swamps. Extinction reached a peak in the Upper Barremian, coinciding with the marine flooding of freshwater environments in Mesogea, and continued through the Aptian and Albian due to substitution of carbonatic freshwater swamps by terrigenous deltaic environments and probably by development of highly competitive aquatic flora of angiosperms. Anagenetic change within species attained maxima during the Berriasian and Lower Barremian, when freshwater environments became extensively developed. Absence of change (stasis) was marked during the Valanginian and Hauterivian, in a geological context of environmental stability and areal reduction of the freshwater environments.