guanosine-triphosphate and 7-methylguanosine-triphosphate

We report synthesis and properties of a pair of new potent inhibitors of translation, namely two diastereomers of 7-methylguanosine 5'-(1-thiotriphosphate). These new analogs of mRNA 5'cap (referred to as m(7)GTPalphaS (D1) and (D2)) are recognized by translational factor eIF4E with high affinity and are not susceptible to hydrolysis by Decapping Scavenger pyrophosphatase (DcpS). The more potent of diastereomers, m(7)GTPalphaS (D1), inhibited cap-dependent translation in rabbit reticulocyte lysate approximately 8-fold and approximately 15-fold more efficiently than m(7)GTP and m(7)GpppG, respectively. Both analogs were also significantly more stable in RRL than unmodified ones.

Topics: Animals; Endoribonucleases; Eukaryotic Initiation Factor-4E; Guanosine Triphosphate; Humans; Mice; Protein Biosynthesis; Protein Synthesis Inhibitors; RNA Cap Analogs; RNA Caps; RNA, Messenger; Stereoisomerism; Thionucleotides

Electric charge distribution in mRNA 5' cap terminus has been exhaustively characterized in respect to the affinity for cap-binding proteins. Formation of the stacked configuration of positively charged 7-methylguanine in between two aromatic amino acid rings, known as sandwich cation-pi stacking, is thought to be prerequisite for the specific recognition of the cap by eukaryotic initiation factor eIF4E; i.e., discrimination between the cap and nucleotides without the methyl group at N(7). Nuclear magnetic resonance spectroscopy of (15)N/(13)C-double-labeled 7-methylguanosine 5'-triphosphate and 7-methylguanosine, as well as their unsubstituted counterparts, GTP and guanosine, yielded characteristic changes of the electron-mediated spin-spin couplings and chemical shifts due to the methylation at N(7). The experimentally measured changes of the nuclear magnetic resonance parameters have been analyzed in respect to the electric charge distribution calculated by means of quantum chemical methods, and interpreted in terms of new proposed positive charge localization in the 7-methylguanine five-member ring.

Topics: Biophysical Phenomena; Biophysics; Carbon; Cations; Electrons; Eukaryotic Initiation Factor-4E; Guanine; Guanosine; Guanosine Triphosphate; Hydrogen-Ion Concentration; Magnetic Resonance Spectroscopy; Methylation; Models, Chemical; RNA Cap Analogs; RNA Caps; RNA, Messenger; Temperature

The Semliki Forest virus (SFV) replicase protein nsP1 has methyltransferase (MT) and guanylyltransferase-like (GT) activities, which are involved in the capping of viral mRNAs. MT catalyzes the transfer of the methyl group from S-adenosylmethionine (AdoMet) to position 7 of GTP, and this reaction is followed by GT-catalyzed formation of the covalent complex m7GMP-nsP1. These reactions are virus specific and thus potential targets for inhibitors of virus replication. We have mutated residues of SFV nsP1, which are conserved in related proteins of the large alphavirus-like superfamily. Mutations of D64, D90, R93, C135, C142, and Y249 to alanine destroyed or greatly reduced the MT activity of nsP1. All MT-negative mutants lost also the GT activity, confirming that methylation of GTP is an essential prerequisite for the synthesis of the covalent guanylate complex. Mutation of H38 prevented the GT reaction without destroying MT activity. Conservation of residues essential for both reactions in the alphavirus-like superfamily implies that they use a capping mechanism similar to that for the alphaviruses. Residues D64 and D90 were necessary for AdoMet binding, as measured by UV cross-linking. Secondary structure predictions of nsP1 and other proteins of the superfamily place these residues in positions corresponding to AdoMet-binding sites of cellular methyltransferases, suggesting that they all may be structurally related.

Topics: Amino Acid Sequence; Cross-Linking Reagents; Gene Expression; Guanosine Triphosphate; HeLa Cells; Humans; Methyltransferases; Molecular Sequence Data; Nucleotidyltransferases; Point Mutation; RNA Cap Analogs; RNA Caps; RNA, Viral; S-Adenosylmethionine; Semliki forest virus; Substrate Specificity; Viral Proteins

The methylation of the 5' terminal guanosine residue of the cap structure of Semliki Forest virus (SFV) mRNAs has been shown to occur in vitro concomitantly with their synthesis (R. K. Cross and P. J. Gomatos, Virology, 114, 542-554, 1981). The enzyme responsible for this methylation, a guanine-7-methyltransferase, is associated with the SFV replication complex which contains both the virus-specified polymerase and RNA template in a mitochondrial pellet fraction, P-15, from infected cell lysates. In the present report, evidence has been obtained demonstrating that a virus-specified function is required for this methylating activity. First, the methyltransferase enzyme in these infected P-15 extracts has been found to differ in substrate specificity from that of the BHK host cell enzyme. This enzyme was able to catalyze the methylation of GTP to m7GTP in vitro whereas the cellular enzyme could not methylate GTP. The incorporation of a methyl group onto GTP occurred linearly for at least 2 hr at 30 degrees under conditions of neutral pH and added GTP substrate. Second, a study of the kinetics of appearance of this activity, has demonstrated that the capacity to methylate GTP did not appear until 1 hr after infection and reached maximal levels by about 3 hr. Third, de novo protein synthesis was required. Addition of the protein synthesis inhibitor, cycloheximide, prevented the appearance and subsequent increase in the methylating activity. However, once formed the methyltransferase was found to be stable for at least 3 hr. These results suggest that an early viral function, perhaps a nonstructural polypeptide is required for this novel guanine-7-methyltransferase activity in SFV infected cell extracts.

Topics: Animals; Cell Extracts; Cell Line; Cell Nucleus; Cricetinae; Cycloheximide; DNA-Directed RNA Polymerases; Guanosine Triphosphate; Hydrogen-Ion Concentration; Kinetics; Methylation; Methyltransferases; Mitochondria; RNA Cap Analogs; Semliki forest virus; Substrate Specificity; Transcription, Genetic

Topics: Animals; Cytidine Triphosphate; Globins; Guanosine Triphosphate; Kinetics; Mathematics; Protein Biosynthesis; Rabbits; Reticulocytes; Ribosomes; RNA Cap Analogs; RNA, Messenger