Detecting allosteric sites of HIV-1 reverse transcriptase by X-ray crystallographic fragment screening.

HIV-1 reverse transcriptase (RT) undergoes a series of conformational changes during viral replication and is a central target for antiretroviral therapy. The intrinsic flexibility of RT can provide novel allosteric sites for inhibition. Crystals of RT that diffract X-rays to better than 2 Å resolution facilitated the probing of RT for new druggable sites using fragment screening by X-ray crystallography.

Cap and cap-binding proteins in the control of gene expression.

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.

Structure of the guanylyltransferase domain of human mRNA capping enzyme.

The enzyme guanylyltransferase (GTase) plays a central role in the three-step catalytic process of adding an (m7)GpppN cap cotranscriptionally to nascent mRNA (pre-mRNAs). The 5'-mRNA capping process is functionally and evolutionarily conserved from unicellular organisms to human. However, the GTases from viruses and yeast have low amino acid sequence identity (∼25%) with GTases from mammals that, in contrast, are highly conserved (∼98%).

3D jigsaw puzzle in rotavirus assembly.

In this issue of Structure, Lu et al. (2008) report results of structural and functional analysis of rotavirus RNA-dependent RNA polymerase, VP1. Based on their analyses of VP1 in RNA free and bound forms, the authors propose a mechanism for coordinated genome packaging and replication.

Species-specific cis-regulatory elements in the 3'-untranslated region direct alternative polyadenylation of bone morphogenetic protein 2 mRNA.

BMP2 (bone morphogenetic protein 2) is a multifunctional member of the transforming growth factor-beta family of growth factors. Disruption of BMP2 signaling results in developmental defects, cancers, and other diseases. BMP2 mRNAs are alternatively polyadenylated, resulting in mRNAs with distinct 3'-untranslated regions.

Apoptosis and autophagy induction in mammalian cells by small interfering RNA knockdown of mRNA capping enzymes.

Addition of a 5' cap to RNA polymerase II transcripts, the first step of pre-mRNA processing in eukaryotes from yeasts to mammals, is catalyzed by the sequential action of RNA triphosphatase, guanylyltransferase, and (guanine-N-7)methyltransferase. The effects of knockdown of these capping enzymes in mammalian cells were investigated using T7 RNA polymerase-synthesized small interfering RNA and also a lentivirus-based inducible, short hairpin RNA system. Decreasing either guanylyltransferase or methyltransferase resulted in caspase-3 activation and elevated terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) staining characteristic of apoptosis.

Crystal engineering of HIV-1 reverse transcriptase for structure-based drug design.

HIV-1 reverse transcriptase (RT) is a primary target for anti-AIDS drugs. Structures of HIV-1 RT, usually determined at approximately 2.5-3.

High-resolution structures of HIV-1 reverse transcriptase/TMC278 complexes: strategic flexibility explains potency against resistance mutations.

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 ( approximately 25-75 mg/day). We have determined the structure of wild-type HIV-1 RT complexed with TMC278 at 1.8 A resolution, using an RT crystal form engineered by systematic RT mutagenesis.

Human capping enzyme promotes formation of transcriptional R loops in vitro.

Cap formation is the first step of pre-mRNA processing in eukaryotic cells. Immediately after transcription initiation, capping enzyme (CE) is recruited to RNA polymerase II (Pol II) by the phosphorylated carboxyl-terminal domain of the Pol II largest subunit (CTD), allowing cotranscriptional capping of the nascent pre-mRNA. Recent studies have indicated that CE affects transcription elongation and have suggested a checkpoint model in which cotranscriptional capping is a necessary step for the early phase of transcription.

Human mRNA cap methyltransferase: alternative nuclear localization signal motifs ensure nuclear localization required for viability.

A characteristic feature of gene expression in eukaryotes is the addition of a 5'-terminal 7-methylguanine cap (m7GpppN) to nascent pre-mRNAs in the nucleus catalyzed by capping enzyme and cap methyltransferase. Small interfering RNA (siRNA) knockdown of cap methyltransferase in HeLa cells resulted in apoptosis as measured by terminal deoxynucleotidyltransferase-mediated dUTP-tetramethylrhodamine nick end labeling assay, demonstrating the importance of mRNA 5'-end methylation for mammalian cell viability. Nuclear localization of cap methyltransferase is mediated by interaction with importin-alpha, which facilitates its transport and selective binding to transcripts containing 5'-terminal GpppN.

Functional interactions of RNA-capping enzyme with factors that positively and negatively regulate promoter escape by RNA polymerase II.

Capping of the 5' ends of nascent RNA polymerase II transcripts is the first pre-mRNA processing event in all eukaryotic cells. Capping enzyme (CE) is recruited to transcription complexes soon after initiation by the phosphorylation of Ser-5 of the carboxyl-terminal domain of the largest subunit of RNA polymerase II. Here, we analyze the role of CE in promoter clearance and its functional interactions with different factors that are involved in promoter clearance.

The Tat/TAR-dependent phosphorylation of RNA polymerase II C-terminal domain stimulates cotranscriptional capping of HIV-1 mRNA.

The HIV type 1 (HIV-1) Tat protein stimulates transcription elongation by recruiting P-TEFb (CDK9/cyclin T1) to the transactivation response (TAR) RNA structure. Tat-induced CDK9 kinase has been shown to phosphorylate Ser-5 of RNA polymerase II (RNAP II) C-terminal domain (CTD). Results presented here demonstrate that Tat-induced Ser-5 phosphorylation of CTD by P-TEFb stimulates the guanylyltransferase activity of human capping enzyme and RNA cap formation.

mRNA capping enzyme requirement for Caenorhabditis elegans viability.

Capping of the initiated 5' ends of RNA polymerase II products is evolutionarily and functionally conserved from yeasts to humans. The m(7)GpppN cap promotes RNA stability, processing, transport, and translation. Deletion of capping enzymes in yeasts was shown to be lethal due to rapid exonucleolytic degradation of uncapped transcripts or failure of capped but unmethylated RNA to initiate protein synthesis.

Cap methyltransferase selective binding and methylation of GpppG-RNA are stimulated by importin-alpha.

We screened a human cDNA library for proteins that bind mRNA cap methyltransferase (MT) and isolated nuclear transporter importin-alpha (Impalpha). This direct association was confirmed by glutathione S-transferase (GST) pulldown, coimmunoprecipitation, and nuclear colocalization. In gel shift assays, MT selectively bound RNA containing 5'-terminal GpppG, and binding was inhibited by GpppG and not by m(7)GpppC.

Viral and cellular mRNA capping: past and prospects.

This chapter focuses on the history of the discovery of cap and an update of research on viral and cellular-messenger RNA (mRNA) capping. Cap structures of the type m GpppN(m)pN(m)p are present at the 5′ ends of nearly all eukaryotic cellular and viral mRNAs. A cap is added to cellular mRNA precursors and to the transcripts of viruses that replicate in the nucleus during the initial phases of transcription and before other processing events, including internal NA methylation, 3′-poly (A) addition, and exon splicing.

The ends of the affair: capping and polyadenylation.

Nearly all mRNAs are post-transcriptionally modified at their 5' and 3' ends, by capping and polyadenylation, respectively. These essential modifications are of course chemically quite distinct, as are the enzymatic complexes responsible for their synthesis. But recent studies have uncovered some similarities as well.

Transcription elongation factor hSPT5 stimulates mRNA capping.

RNA polymerase II nascent transcripts are capped during pausing before elongation. Here we report that hSPT5, the human homolog of yeast elongation factor SPT5, interacts directly with the capping enzyme. hSPT5 stimulated capping enzyme guanylylation and mRNA capping by severalfold.

Genomic structure and chromosomal localization of TCEAL1, a human gene encoding the nuclear phosphoprotein p21/SIIR.

Human p21/SIIR is a novel Ser/Arg/Pro-rich nuclear phosphoprotein that is 48% similar to transcription factor SII and modulates transcription in a promoter context-dependent fashion. We have obtained the complete sequence of TCEAL1, the gene that codes for p21/SIIR. This gene consists of three exons and two introns with the entire coding sequence in exon III.

Mammalian capping enzyme binds RNA and uses protein tyrosine phosphatase mechanism.

Mammalian capping enzymes are bifunctional proteins with both RNA 5'-triphosphatase and guanylyltransferase activities. The N-terminal 237-aa triphosphatase domain contains (I/V)HCXXGXXR(S/T)G, a sequence corresponding to the conserved active-site motif in protein tyrosine phosphatases (PTPs). Analysis of point mutants of mouse RNA 5'-triphosphatase identified the motif Cys and Arg residues and an upstream Asp as required for activity.

Recombinant human mRNA cap methyltransferase binds capping enzyme/RNA polymerase IIo complexes.

Guanine N-7 methylation is an essential step in the formation of the m7GpppN cap structure that is characteristic of eukaryotic mRNA 5' ends. The terminal 7-methylguanosine is recognized by cap-binding proteins that facilitate key events in gene expression including mRNA processing, transport, and translation. Here we describe the cloning, primary structure, and properties of human RNA (guanine-7-)methyltransferase.

Mammalian capping enzyme complements mutant Saccharomyces cerevisiae lacking mRNA guanylyltransferase and selectively binds the elongating form of RNA polymerase II.

5'-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.

Double-stranded RNA-dependent protein kinase (PKR) is regulated by reovirus structural proteins.

Reovirus sigma3 is a virion outer shell protein that also binds dsRNA and stimulates translation by blocking activation of the dsRNA-dependent protein kinase, PKR. Purified sigma3 was shown by gel shift assay to bind specifically to RNA duplexes of minimal length 32-45 base pairs. PKR binding to dsRNA was prevented by sigma3, and translation inhibition of luciferase reporter by PKR expression in transfected cells was reversed by sigma3.

[Immunodiagnostic preparations based on monoclonal antibodies to Chlamydia trachomatis].

Highly sensitive diagnostic preparations for the detection of C. trachomatis by direct immunofluorescent and enzyme immunoassay techniques were obtained with the use monoclonal antibodies to C. trachomatis genus-specific polysaccharide antigen.