ABSTRACT
m(7)GTP-Sepharose is routinely used for cap binding protein isolation. Here we present the synthesis of a new affinity resin containing a mononucleotide cap analog resistant to hydrolysis by DcpS. The resin has been designed in order to identify and purify Arabidopsis thaliana DcpS and other pyrophosphatases. The binding efficiency of the new resin to eIF4E protein was compared with standard m(7)GTP-Sepharose. The utility of non-hydrolysable resin was demonstrated on yeast extract.
Subject(s)
Chromatography, Affinity , Diphosphonates/chemistry , Pyrophosphatases/isolation & purification , RNA Cap Analogs/chemical synthesis , RNA Cap-Binding Proteins/isolation & purification , Sepharose/analogs & derivatives , Animals , Arabidopsis Proteins/isolation & purification , Chromatography, Agarose , Eukaryotic Initiation Factor-4E/isolation & purification , Mice , RNA Cap Analogs/chemistry , Sepharose/chemical synthesis , Sepharose/chemistryABSTRACT
All eukaryotic mRNAs possess a 5'-cap (m(7)GpppN) that is recognized by a family of cap-binding proteins. These participate in various processes, such as RNA transport and stabilization, as well as in assembly of the translation initiation complex. The 5'-cap of trypanosomatids is complex; in addition to 7-methyl guanosine, it includes unique modifications on the first four transcribed nucleotides, and is thus denoted cap-4. Here we analyze a cap-binding protein of Leishmania, in an attempt to understand the structural features that promote its binding to this unusual cap. LeishIF4E-1, a homolog of eIF4E, contains the conserved cap-binding pocket, similar to its mouse counterpart. The mouse eIF4E has a higher K(as) for all cap analogs tested, as compared with LeishIF4E-1. However, whereas the mouse eIF4E shows a fivefold higher affinity for m(7)GTP than for a chemically synthesized cap-4 structure, LeishIF4E-1 shows similar affinities for both ligands. A sequence alignment shows that LeishIF4E-1 lacks the region that parallels the C terminus in the murine eIF4E. Truncation of this region in the mouse protein reduces the difference that is observed between its binding to m(7)GTP and cap-4, prior to this deletion. We hypothesize that variations in the structure of LeishIF4E-1, possibly also the absence of a region that is homologous to the C terminus of the mouse protein, promote its ability to interact with the cap-4 structure. LeishIF4E-1 is distributed in the cytoplasm, but its function is not clear yet, because it cannot substitute the mammalian eIF4E in a rabbit reticulocyte in vitro translation system.
Subject(s)
Eukaryotic Initiation Factor-4E/metabolism , Guanosine Diphosphate/analogs & derivatives , Guanosine Diphosphate/metabolism , Leishmania/metabolism , RNA Cap-Binding Proteins/metabolism , Amino Acid Sequence , Animals , Cells, Cultured , Computer Simulation , Conserved Sequence , Cytoplasm/chemistry , Eukaryotic Initiation Factor-4E/chemistry , Fluorescent Antibody Technique, Indirect , Guanosine Diphosphate/chemistry , Kinetics , Leishmania major/metabolism , Microscopy, Fluorescence , Models, Molecular , Molecular Sequence Data , Protein Structure, Secondary , RNA Cap-Binding Proteins/isolation & purification , RNA, Protozoan/isolation & purification , RNA, Protozoan/metabolism , Recombinant Proteins/metabolism , Sequence Homology, Amino AcidABSTRACT
The nuclear import of spliceosomal UsnRNPs is mediated by the transport adaptor snurportin 1 (SPN1), which specifically recognizes the 2,2,7-trimethylguanosine (m(3)G) cap at the 5' end of UsnRNAs. Human SPN1 was overexpressed as a GST-fusion protein in Escherichia coli and purified to homogeneity. Since full-length SPN1 did not crystallize, limited proteolysis experiments were performed and stable digestion products were analyzed for functionality with respect to m(3)G cap-binding activity and subsequently used for crystallization trials. Well diffracting single crystals of a truncated SPN1 m(3)G cap-binding domain (residues 79-300) were obtained after two rounds of seeding. The crystals belong to space group P4(1)2(1)2 or P4(3)2(1)2, with unit-cell parameters a = b = 57.47, c = 130.09 A, alpha = beta = gamma = 90 degrees. Crystals contain one molecule in the asymmetric unit and diffract to a resolution limit of 2.9 A.
