TMC278 is a diarylpyrimidine (DAPY) nonnucleoside reverse transcriptase inhibitor (NNRTI) that is highly effective in treating wild-type and drug-resistant HIV-1 infections in clinical trials at relatively low doses (ϳ25-75 mg/day). We have determined the structure of wild-type HIV-1 RT complexed with TMC278 at 1.8 Å resolution, using an RT crystal form engineered by systematic RT mutagenesis. This highresolution structure reveals that the cyanovinyl group of TMC278 is positioned in a hydrophobic tunnel connecting the NNRTI-binding pocket to the nucleic acid-binding cleft. The crystal structures of TMC278 in complexes with the double mutant K103N/Y181C (2.1 Å) and L100I/K103N HIV-1 RTs (2.9 Å) demonstrated that TMC278 adapts to bind mutant RTs. In the K103N/Y181C RT/TMC278 structure, loss of the aromatic ring interaction caused by the Y181C mutation is counterbalanced by interactions between the cyanovinyl group of TMC278 and the aromatic side chain of Y183, which is facilitated by an ϳ1.5 Å shift of the conserved Y183MDD motif. In the L100I/K103N RT/ TMC278 structure, the binding mode of TMC278 is significantly altered so that the drug conforms to changes in the binding pocket primarily caused by the L100I mutation. The flexible binding pocket acts as a molecular ''shrink wrap'' that makes a shape complementary to the optimized TMC278 in wild-type and drug-resistant forms of HIV-1 RT. The crystal structures provide a better understanding of how the flexibility of an inhibitor can compensate for drug-resistance mutations.drug design ͉ drug resistance ͉ nonnucleoside reverse transcriptase inhibitor ͉ polymerase ͉ x-ray crystallography C ombinations of drugs have been used to successfully treat HIV-1 infections, permitting dramatic reduction in viral loads and restoration of the immune system. However, drug treatments do not eliminate the infection and treatment must be life-long. There are problems with drug toxicity, and prolonged drug exposure can lead to the emergence of drug-resistant mutant viruses. Because toxicity and drug resistance complicate treatment strategies, the development of new anti-AIDS drugs remains essential., also referred to as rilpivirine and R278474, is a diarylpyrimidine (DAPY) nonnucleoside reverse transcriptase inhibitor (NNRTI) that was developed in a multidisciplinary structure-based design effort (3, 4) intended to discover drugs that can inhibit a wide range of the known drug-resistant HIV-1 strains. In cell-based assays, TMC278 inhibits a broad spectrum of HIV-1 variants (Table 1), including strains that are partially or completely resistant to the existing NNRTI drugs (3). Phase I and II clinical trials have shown that TMC278 effectively reduces viral load and maintains low viral load levels with no virus rebound in patients even after 48 weeks of treatment (5, 6).Crystal structures of HIV-1 RT/NNRTI complexes were solved in the course of developing TMC278 (7, 8) along a path that originated with the first-generation TIBO compounds (9). Most of those structures were obtained at ϳ...
The 5' mRNA cap structure is essential for efficient gene expression from yeast to human. It plays a critical role in all aspects of the life cycle of an mRNA molecule. Capping occurs co-transcriptionally on the nascent pre-mRNA as it emerges from the RNA exit channel of RNA polymerase II. The cap structure protects mRNAs from degradation by exonucleases and promotes transcription, polyadenylation, splicing, and nuclear export of mRNA and U-rich, capped snRNAs. In addition, the cap structure is required for the optimal translation of the vast majority of cellular mRNAs, and it also plays a prominent role in the expression of eukaryotic, viral, and parasite mRNAs. Cap-binding proteins specifically bind to the cap structure and mediate its functions in the cell. Two major cellular cap-binding proteins have been described to date: eukaryotic translation initiation factor 4E (eIF4E) in the cytoplasm and nuclear cap binding complex (nCBC), a nuclear complex consisting of a cap-binding subunit cap-binding protein 20 (CBP 20) and an auxiliary protein cap-binding protein 80 (CBP 80). nCBC plays an important role in various aspects of nuclear mRNA metabolism such as pre-mRNA splicing and nuclear export, whereas eIF4E acts primarily as a facilitator of mRNA translation. In this review, we highlight recent findings on the role of the cap structure and cap-binding proteins in the regulation of gene expression. We also describe emerging regulatory pathways that control mRNA capping and cap-binding proteins in the cell.
Reovirus mRNAs with 5'terminal m7GpppGm or GpppG are more stable than mRNA containing unblocked ppG 5'-ends when injected into Xenopus laevis oocytes or incubated in cell-free protein synthesising extracts of wheat germ and mouse L cells. The greater stability of mRNA with blocked 5' termini is not dependent upon translation but seems to result from protection against 5'-exonucleolytic degradation.
ABSTRACT5-Capping is an early mRNA modification that has important consequences for downstream events in gene expression. We have isolated mammalian cDNAs encoding capping enzyme. They contain the sequence motifs characteristic of the nucleotidyl transferase superfamily. The predicted mouse and human enzymes consist of 597 amino acids and are 95% identical. Mouse cDNA directed synthesis of a guanylylated 68-kDa polypeptide that also contained RNA 5-triphosphatase activity and catalyzed formation of RNA 5-terminal GpppG. A haploid strain of Saccharomyces cerevisiae lacking mRNA guanylyltransferase was complemented for growth by the mouse cDNA. Conversion of Lys-294 in the KXDG-conserved motif eliminated both guanylylation and complementation, identifying it as the active site. The K294A mutant retained RNA 5-triphosphatase activity, which was eliminated by N-terminal truncation. Full-length capping enzyme and an active C-terminal fragment bound to the elongating form and not to the initiating form of polymerase. The results document functional conservation of eukaryotic mRNA guanylyltransferases from yeast to mammals and indicate that the phosphorylated C-terminal domain of RNA polymerase II couples capping to transcription elongation. These results also explain the selective capping of RNA polymerase II transcripts.Addition of a 5Ј-terminal cap is an important, early event in mRNA formation (1). This structural hallmark of most eukaryotic mRNAs enhances splicing (2-4), transport (5), translation (6), and stability (7,8) and is essential for viability (9).Caps are formed on nascent nuclear pre-mRNAs by conversion of 5Ј-tri-diphosphate to 5Ј-diphosphate ends, followed by addition of GMP and methylation (1, 10). The guanylyltransfer reaction characterized in various systems involves formation of an active enzyme intermediate containing GMP covalently attached to lysine (11). In yeast, mRNA capping enzyme consists of separate subunits for RNA 5Ј-triphosphatase and guanylyltransferase activities (9, 12). cDNA clones coding for mRNA guanylyltransferase in Saccharomyces cerevisiae (9), Schizosaccharomyces pombe (13), and Candida albicans (14) have been sequenced. Each contains the active site lysine in KXDG (13, 15), one of several highly conserved motifs characteristic of a superfamily of nucleotidyl transferases (16). A number of viral capping enzymes also contain these diagnostic sequence motifs, and the recently solved structure of capping enzyme from Chlorella virus PBCV-1 suggests that specific residues in these motifs are important for binding GTP (17). Despite this detail of sequence and structure information, no metazoan capping enzyme previously has been cloned and characterized.To explore the molecular interactions that result in selective capping of RNA polymerase II (pol II) transcripts in mammalian cells, we have isolated and characterized cDNA clones that code for the human and mouse capping enzymes. Functional studies demonstrated that the mammalian enzyme complements the lethality of a S. cerevisiae mu...
Protein synthesis initiation factors prepared from rabbit reticulocyte and mouse ascites ribosomes were tested for the ability to crosslink to the 5' cap of mRNA. Crosslinking of one polypeptide of apparent molecular weight 24,000 was inhibited by the cap analogs, m7GMP and m7GDP, indicating a specific interaction with the cap. Although specific crosslinking of the 24,000 molecular weight polypeptide was found with eukaryotic initiation factor 3 and to a lesser extent with initiation factor 4B, both of these factors contained less than stoichiometric amounts of this polypeptide. The crosslinking method provides a highly sensitive and specific assay for cap-binding proteins and should facilitate their purification for functional studies.
Poly(A)-containing HeLa cell mRNA prepared from cells labeled with [3Plphosphate was found to contain a variety of methylated, blocked 5'-terminal structures of two general types: m7GpppNm-Np and m7GpppNm-Nm-Np. In addition, about one-third of the [3Hlmethyl label was present in the N6-methyladenosine; this labeled nucleoside was not found in the 3'-terminal one-third of the mRNA chain and thus may also be in the 5' portion of the mRNA.The 5' end of a variety of viral mRNA molecules consists of an unusual type of methylated oligonucleotide with the general structure m7G(5')ppp(5')Nm-Np (1-6). Because the addition through pyrophosphate linkage of the terminal m7G renders the terminal dinucleotide resistant to digestion by the usual ribonucleases, the structure has been called a "cap" (7). Perry and Kelley (8) have shown that mammalian cell mRNA contains methyl groups, and, with the availability of methods used to characterize the cap structures in viral mRNAs, we have examined HeLa cell mRNA for the presence of caps. As pointed out by Rottman et al. (7), information about various steps of methylation of potential mRNA precursors by the cell should aid in understanding eukaryotic mRNA formation. MATERIALS AND METHODSRadioactive Labeling. Growth of suspension cultures of HeLa cells was in Eagle's medium. Cells were labeled with 32p for 4 hr in phosphate-free medium as described (9). Labeling with [methyl-3H]
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.