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At least 145 records · Page 8Linked to original sources

PubMed2002

Loss of activities for mRNA synthesis accompanies loss of lambda2 spikes from reovirus cores: an effect of lambda2 on lambda1 shell structure.

The 144-kDa lambda2 protein, a component of the transcriptionally active reovirus core particle, catalyzes the last three enzymatic activities for formation of the 5' cap 1 structure on the viral plus-strand transcripts. Limited evidence suggests it may also play a role in transcription per se. Particle-associated lambda2 forms pentameric turrets ("spikes") around the fivefold axes of the icosahedral core. To address the requirements for lambda2 in core functions other than the known functions in RNA capping, particles depleted of lambda2 were generated from cores in vitro by a series of treatments involving heat, protease, and ionic detergent. The resulting particles contained less than 5% of pretreatment levels of lambda2 but showed negligible loss of the other four core proteins or the 10 double-stranded RNA genome segments. Transmission cryo-electron microscopy (cryo-TEM) and scanning cryo-electron microscopy demonstrated loss of the lambda2 spikes from these otherwise intact particles. In functional analyses, the "spikeless cores" showed greatly reduced activities not only for RNA capping but also for transcription and nucleoside triphosphate hydrolysis, suggesting enzymatic or structural roles for lambda2 in all these activities. Comparison of the core and spikeless core structures obtained by cryo-TEM and three-dimensional image reconstruction revealed changes in the lambda1 core shell that accompany lambda2 loss, most notably the elimination of small pores that span the shell near the icosahedral fivefold axes. Changes in the shell may explain the reductions in transcriptase-related activities by spikeless cores.

Cryoelectron Microscopy↗

PubMed1982

A polypeptide which reverses cap analogue inhibition of cell-free protein synthesis. Purification and binding to capped oligonucleotides.

An assay was developed to detect the component which recognizes the methylated 5'-terminus of messenger RNA (cap) during initiation of translation. Globin mRNA translation in a reticulocyte cell-free system was partially inhibited with cap analogues, and protein fractions were added to the system in an attempt to reverse inhibition. Such an activity was detected in the 500 mM KCl extract of rabbit reticulocyte ribosomes. The activity (cap analogue inhibition reversal) was purified 800-fold by precipitation with ammonium sulfate saturation, batchwise chromatography on DEAE-cellulose, centrifugation on sucrose gradients containing 100 and 500 mM KCl, and column chromatography on DEAE-cellulose and phosphocellulose. At some stages multiple peaks of activity were detected. Electrophoretic analysis of the final preparation revealed a single polypeptide of 24,000 daltons, making it likely that it is the same as the cap-binding protein detected by Sonenberg et al. (Sonenberg, N., Morgan, M. A., Merrick, W. C., and Shatkin, A. J. (1978) Proc. Natl. Acad. Sci. U. S. A. 75, 4843-4847), using a cross-linking assay. Direct binding of purified fractions of cap analog inhibition reversal factor to capped oligonucleotides of the form m7Gppp(Np)6-8G[32P]Cp could be demonstrated by both gel filtration on Sephadex G-50 and nitrocellulose membrane filtration. Binding was stimulated by MgCl2 and nucleoside triphosphates. An approximate association constant between oligonucleotide and protein of 3 X 10(8) M-1 was obtained.

Animals↗

PubMed1998

Guanylyltransferase and RNA 5'-triphosphatase activities of the purified expressed VP4 protein of bluetongue virus.

We have examined the RNA-capping enzyme activities of bluetongue virus (BTV) minor core protein, VP4. Recombinant BTV VP4 protein was purified to homogeneity from insect cell culture infected with a baculovirus VP4 of BTV serotype 10. We demonstrate that the purified protein, and VP4 encapsidated in core-like particles, react with GTP and covalently bind GMP via a phosphoamide linkage, a characteristic feature of guanylyltransferase enzyme. VP4 also catalyses a GTP-PPi exchange reaction indicating that the protein is the guanylyltransferase of the virus. In addition, VP4 possesses an RNA 5'-triphosphatase activity which catalyses the first step in the RNA-capping sequence. Further, an inorganic pyrophosphatase activity was identified which may aid the transcription activity within the virus by removing inorganic pyrophosphate which is an inhibitor of the polymerization reaction. Finally, the direct evidence of VP4 capping activity has been obtained by demonstrating in vitro transfer of GMP to the 5' end of in vitro synthesized BTV ssRNA transcripts to form a cap structure.

Acid Anhydride Hydrolases↗

PubMed1994

Functional analysis of glucocorticoid and insulin response sequences in the rat insulin-like growth factor-binding protein-1 promoter.

Insulin-like growth factor-binding protein-1 (IGFBP-1) is produced by the liver and regulated by glucocorticoids and insulin at the level of gene transcription. To identify DNA sequences mediating the effects of glucocorticoids and insulin on IGFBP-1 promoter activity we created luciferase reporter gene constructs and performed transfection studies in H4IIE hepatoma cells. Initial studies confirmed that the IGFBP-1 promoter is functional when inserted in the correct orientation, but not in the reverse orientation. Dexamethasone (DEX) increased promoter activity 10-fold, and insulin reversed this effect of DEX by 85% at 8 h. The effects of DEX were abolished when constructs were truncated to 89 bases from the RNA cap site, and DNase footprinting with the DNA-binding domain of the human glucocorticoid receptor identified an imperfect palindrome containing two receptor-binding sites separated by three nucleotides typical of a glucocorticoid response element (GRE) at this location. Mutation of either binding site (or half-site) disrupted the effects of DEX, confirming that this sequence functions as a GRE. Two other regions of the promoter also footprinted with the glucocorticoid receptor protein and contained sequences consistent with glucocorticoid receptor-binding sites; however, neither of these footprints contained the full structure expected of a functional GRE, and neither mutation nor deletion of these other sequences altered the effects of DEX on promoter activity. To identify the DNA sequences required for the effects of insulin on glucocorticoid-stimulated promoter activity, we created internal deletions throughout the IGFBP-1 promoter region. Deletion of the 22-basepair (bp) sequence immediately 5' from the GRE disrupted the effect of insulin and appeared to increase basal promoter activity at least 2-fold in each of eight experiments (P < 0.001 vs. intact promoter). This region of the IGFBP-1 promoter contains a 19-bp palindrome (CAAAACAAACTTATTTTG) that overlaps the 5'-end of the GRE and is fully conserved in the human IGFBP-1 promoter. Each half of this palindrome resembles previously identified insulin response sequences, and deletion/mutation analysis suggests that each half may contribute to the effects of insulin on promoter activity. Gel shift studies confirmed that this palindrome binds H4IIE nuclear proteins. In summary, we have identified a GRE in the 5'-promoter region of the rat IGFBP-1 gene approximately 90 bp up-stream from the RNA cap site as well as a contiguous 22-bp region that plays a critical role in mediating the effects of insulin on glucocorticoid-stimulated promoter activity.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed2006

Comparison of promoters controlling on the sunflower mitochondrial genome the transcription of two copies of the same native trnK gene reveals some differences in their structure.

Two copies of native trnK (UUU) gene are encoded on the sunflower mitochondrial DNA. They lie within two 12-kb direct repeats, presumably generated by a duplication event. During an investigation aimed at detecting DNA regions activating the trnK1 and trnK2 genes, three distinct promoters have been identified. Their locations were deduced using standard procedures (RT-PCR, RNA capping and 5'RACE) usually employed for the detection of transcription initiation sites (TISs). Promoters P3 and P2 control two independent partially overlapping transcription units containing the trnK2 and ccb206 genes, respectively. Promoter P1 has been mapped about 5200 bp upstream of the trnK1 gene which is part of a transcription unit also containing exons c, d and e of the nad2 gene, 5' to the tRNA gene. Most probably this promoter is not alone in controlling this transcription unit because this DNA region could be cotranscribed, at least partially, starting from other two promoters located upstream of the trnC and trnN genes, respectively. These genes have been previously mapped in a 5' region adjacent to the cluster containing nad2 exons c, d and e and the trnK1 gene. The comparative analysis of promoters P3 and P1 suggests that the difference between them could be related to the duplication event generating the second copy of trnK gene. The availability of a high number of new promoters belonging to dicot mitochondrial genomes makes possible to note some of their specific features.

Base Sequence↗

PubMed1998

Fluorescence quenching and dequenching analysis of RNA interactions in vitro and in vivo.

This paper describes the use of fluorescence quenching and dequenching to analyze molecular interactions of RNA in vitro and in vivo. Fluorescein-labeled ribonucleotide was incorporated into an RNA substrate by in vitro transcription. The fluorescence quantum yield of the intact RNA was reduced by intramolecular quenching. When the RNA was degraded by ribonuclease digestion, the quantum yield increased by approximately 50%, reflecting dequenching due to separation of proximate fluorophores. Dequenching was dependent on the concentration of enzyme and substrate and was inhibited by the ribonuclease inhibitor RNasin. Comparable rates of dequenching were observed with RNase A and RNase T1. Dequenching provides a sensitive, quantitative, and convenient assay for RNA degradation. When fluorescent RNA was microinjected into cells in culture the intracellular fluorescence declined gradually with time after injection reflecting "superquenching: due to intermolecular interactions between the injected RNA and intracellular components. Capped RNA exhibited greater superquenching than uncapped RNA. Superquenching provides a sensitive, quantitative, and specific assay with subcellular resolution for intermolecular interactions of RNA in vivo. When RNase was injected into the same cells, fluorescence increased by approximately 50%, indicating that fluorescence dequenching due to RNA degradation can be measured in vivo as well as in vitro.

Animals↗

PubMed1998

A DNA clone encoding the full-length infectious genome of odontoglossum ringspot tobamovirus and mutagenesis of its coat protein gene.

A full-length DNA clone encoding the genome of odontoglossum ringspot tobamovirus (ORSV) was synthesized and placed adjacent to a bacteriophage T7 RNA polymerase promoter. Capped-RNA transcripts produced in vitro were highly infectious when mechanically inoculated onto seedlings of Nicotiana benthamiana and Oncidium Gower Ramsey. A representative clone, designated pOT2, caused a disease phenotype identical to that produced by parental viral RNA. ELISA, Western blot analysis, Northern blot hybridization and electron microscopy verified the infectivity of pOT2. A coat protein deficient mutant of the clone, pO delta CP1, was produced with the initiation codon of the coat protein cistron of ORSV abolished. Transcripts from pO delta CP1 were infective, able to move in N. benthamiana but produced no coat protein. This demonstrates that the coat protein was dispensable for RNA replication and for movement. This is believed to be the first report of an ORSV infectious clone driven by a T7 RNA polymerase promoter.

Blotting, Northern↗

PubMed2005

Novel synthesis of nucleoside 5'-phosphoramidates through reaction of nucleoside triphosphates with amines mediated by trimethylsilyl chloride.

Reaction of nucleoside triphosphates (NTPs) with amines in pyridine mediated by trimethylsilyl chloride produced nucleoside 5'-phosphoramidates in moderate yields without any preprotection of nucleosides and amino acid methyl esters. The reaction pathway is very similar to the mechanism of the RNA capping reaction, DNA or RNA ligation reaction, and catalysis of hydrolases and nucleases involving the formation of covalent enzyme-NMP (nucleoside 5'-monophosphate) intermediates in biological systems, which could provide a valuable clue for the enzymatic reactions.

Amines↗

PubMed2003

Mutational analysis of bacteriophage T4 RNA ligase 1. Different functional groups are required for the nucleotidyl transfer and phosphodiester bond formation steps of the ligation reaction.

T4 RNA ligase 1 (Rnl1) exemplifies an ATP-dependent RNA ligase family that includes fungal tRNA ligase (Trl1) and a putative baculovirus RNA ligase. Rnl1 acts via a covalent enzyme-AMP intermediate generated by attack of Lys-99 N zeta on the alpha phosphorus of ATP. Mutation of Lys-99 abolishes ligase activity. Here we tested the effects of alanine mutations at 19 conserved positions in Rnl1 and thereby identified 9 new residues essential for ligase activity: Arg-54, Lys-75, Phe-77, Gly-102, Lys-119, Glu-227, Gly-228, Lys-240, and Lys-242. Seven of the essential residues are located within counterparts of conserved nucleotidyltransferase motifs I (99KEDG102), Ia (118SK119), IV (227EGYVA231), and V (238HFKIK242) that comprise the active sites of DNA ligases, RNA capping enzymes, and T4 RNA ligase 2. Three other essential residues, Arg-54, Lys-75 and Phe-77, are located upstream of the AMP attachment site within a conserved domain unique to the Rnl1-like ligase family. We infer a shared evolutionary history and active site architecture in Rnl1 (a tRNA repair enzyme) and Trl1 (a tRNA splicing enzyme). We determined structure-activity relationships via conservative substitutions and examined mutational effects on the isolated steps of Rnl1 adenylylation (step 1) and phosphodiester bond formation (step 3). Lys-75, Lys-240, and Lys-242 were found to be essential for step 1 and overall ligation of 5'-phosphorylated RNA but not for phosphodiester bond formation. These results suggest that the composition of the Rnl1 active site is different during steps 1 and 3. Mutations at Arg-54 and Lys-119 abolished the overall RNA ligation reaction without affecting steps 1 and 3. Arg-54 and Lys-119 are thereby implicated as specific catalysts of the RNA adenylation reaction (step 2) of the ligation pathway.

Alanine↗

PubMed2003

Genetic and biochemical analysis of the functional domains of yeast tRNA ligase.

Yeast tRNA ligase (Trl1) converts cleaved tRNA half-molecules into spliced tRNAs containing a 2'-PO4, 3'-5' phosphodiester at the splice junction. Trl1 performs three reactions: (i) the 2',3'-cyclic phosphate of the proximal fragment is hydrolyzed to a 3'-OH, 2'-PO4 by a cyclic phosphodiesterase (CPD); (ii) the 5'-OH of the distal fragment is phosphorylated by an NTP-dependent polynucleotide kinase; and (iii) the 3'-OH, 2'-PO4, and 5'-PO4 ends are sealed by an ATP-dependent RNA ligase. Trl1 consists of an N-terminal adenylyltransferase domain that resembles T4 RNA ligase 1, a central domain that resembles T4 polynucleotide kinase, and a C-terminal CPD domain that resembles the 2H phosphotransferase enzyme superfamily. Here we show that all three domains are essential in vivo, although they need not be linked in the same polypeptide. We identify five amino acids in the adenylyltransferase domain (Lys114, Glu266, Gly267, Lys284, and Lys286) that are essential for Trl1 activity and are located within motifs I (114KANG117), IV (266EGFVI270), and V (282FFKIK286) that comprise the active sites of DNA ligases, RNA capping enzymes, and T4 RNA ligases 1 and 2. Mutations K404A and T405A in the P-loop (401GXGKT405) of the central kinase-like domain had no effect on Trl1 function in vivo. The K404A and T405A mutations eliminated ATP-dependent kinase activity but preserved GTP-dependent kinase activity. A double alanine mutant in the P-loop was lethal in vivo and abolished GTP-dependent kinase activity. These results suggest that GTP is the physiological substrate and that the Trl1 kinase has a single NTP binding site of which the P-loop is a component. Two other mutations in the central domain were lethal in vivo and either abolished (D425A) or severely reduced (R511A) GTP-dependent RNA kinase activity in vitro. Mutations of the signature histidines of the CPD domain were either lethal (H777A) or conferred a ts growth phenotype (H673A).

Amino Acid Sequence↗

PubMed2005

Differential proteome analysis of aging in rat skeletal muscle.

To identify the mechanisms underlying muscle aging, we have undertaken a high-resolution differential proteomic analysis of gastrocnemius muscle in young adults, mature adults, and old LOU/c/jall rats. Two-dimensional gel electrophoresis and subsequent MALDI-ToF mass spectrometry analyses led to the identification of 40 differentially expressed proteins. Strikingly, most differences characterized old (30-month) animals, whereas young (7-month) and mature (18-month) adults exhibited similar patterns of expression. Important modifications in contractile (actin, myosin light-chains, troponins-T) and cytoskeletal (desmin, tubulin) proteins, and in essential regulatory proteins (gelsolin, myosin binding proteins, CapZ-beta, P23), likely account for dysfunctions in old muscle force generation and speed of contraction. Other features support decreases in cytosolic (triose-phosphate isomerase, enolase, glycerol-3-P dehydrogenase, creatine kinase) and mitochondrial (isocitrate dehydrogenase, cytochrome-c oxidase) energy metabolisms. Muscle aging is often associated with increased oxidative stress. Accordingly, we observed differential regulation of molecular chaperones (hsp20, hsp27, reticuloplasmin ER60) and of proteins implicated in reactive aldehyde detoxification (aldehyde dehydrogenase, glutathione transferase, glyoxalase). We further noticed up-regulation of proteins involved in transcriptional elongation (RNA capping protein) and RNA-editing (Apobec2). Most of these proteins were previously unrecognized as differentially expressed in old muscles, and they represent novel starting points for elucidating the mechanisms of muscle aging.

Aging↗

PubMed2005

Reverse genetics system for porcine enteric calicivirus, a prototype sapovirus in the Caliciviridae.

A porcine enteric calicivirus (PEC), strain Cowden in the genus Sapovirus of the Caliciviridae family, can be propagated in a porcine kidney continuous cell line (LLC-PK) in the presence of bile acids in the cell culture medium. A full-length cDNA copy of the Cowden PEC genome was cloned into a plasmid vector directly downstream from the T7 RNA polymerase promoter, and capped RNA transcripts derived from this clone were infectious when transfected into LLC-PK cells. The recovery of PEC after transfection of RNA transcripts was dependent on the presence of bile acids, consistent with our recent identification of a bile acid-mediated signaling pathway required for PEC replication (Chang et al., Proc. Natl. Acad. Sci. USA 101:8733-8788, 2004). Recovery of virus was verified by detection of PEC antigen in transfected cells by immunofluorescence and enzyme-linked immunosorbent assays, direct observation of recovered viral particles by electron microscopy, and partial sequence analysis of their genomes (first 1,070 nucleotides) to differentiate them from tissue culture-adapted parental virus. The recovered virus retained its ability to infect piglets when administered by the oral route and showed an attenuated phenotype similar to that of the tissue culture-adapted parental virus. This reverse genetics system for PEC provides a new tool to study the molecular basis of replication and pathogenesis for caliciviruses associated with diarrheal disease.

Animals↗

PubMed1997

5'-RNA self-capping from guanosine diphosphate.

A selected RNA (isolate 6) efficiently catalyzes a self-capping reaction with free GDP, yielding the same 5'-capped structure as is formed by protein GTP:RNA guanylyltransferase. This unexplored RNA-catalyzed reaction type involving nucleophilic attack on phosphate by phosphate adds to the variety of possible postsynthetic RNA-catalyzed RNA modifications. The selected RNA requires only Ca2+ for activation and has a broad active pH range of 4.5-9.0. The RNA also has a 5'-pyrophosphatase activity.

Base Sequence↗

PubMed1984

5'-Terminal caps of snRNAs are reactive with antibodies specific for 2,2,7-trimethylguanosine in whole cells and nuclear matrices. Double-label immunofluorescent studies with anti-m3G antibodies and with anti-RNP and anti-Sm autoantibodies.

Antibodies specific for 2,2,7-trimethylguanosine (m3G), which do not cross-react with m7G-capped RNA molecules were used to study, by immunofluorescence microscopy, the reactivity of the m3G-containing cap structures of the snRNAs U1 to U5 in situ. In interphase cells, immunofluorescent sites were restricted to the nucleus, whilst nucleoli were free of fluorescence. This indicates that the 5' terminal of most of the nucleoplasmic snRNAs are not protected by an m3G cap-recognizing protein and that the snRNA caps are not necessarily required for the binding of snRNPs to subnuclear structures. The snRNAs in the nucleoplasm appeared as distinct units in the light microscope, and this allowed the comparison of the distribution of snRNP proteins by double label studies with anti-RNP or anti-Sm antibodies within the same cell. The three antibody classes produced superimposable fluorescent patterns. Taking into account that the various IgGs react with antigenic sites on snRNAs or snRNP proteins not shared by all the snRNP species, these data suggest that U1 snRNP particles are distributed in the same way as the other snRNPs in the nucleus. Qualitatively the same results were obtained with DNase-treated nuclear matrices indicating that intact snRNPs are part of the nuclear matrix. Our data are consistent with proposals that the various snRNPs may be involved in processing of hnRNA and that this may take place at the nuclear matrix.

Animals↗

PubMed2004

Chemical route to the capped RNAs.

Eukaryotic and viral messenger RNAs contain a CAP structure that plays an important role in the initiation of translation and several other cellular processes that involve mRNAs. In this paper, we report a convenient chemical approach to the preparation of milligram quantities of short, capped RNA oligonucleotides, which overcomes some of the limitations of previous approaches. The method is based on the use of a reactive precursor, m7GppQ [P1-7-methylguanosine-5'-O-yl, P2-O-8-(5-chloroquinolyl) pyrophosphate]. The precursor reacts smoothly with 5'-phosphorylated unprotected short RNA in the presence of CuCl2 in organic media. The feasibility of this approach was demonstrated by the synthesis of the capped pentaribonucleotide m7GpppGpApCpU. The synthesized capped oligonucleotide was isolated and purified by reverse phase and ion exchange HPLC with a final yield of 37%. The structure of the m7GpppGpApCpU was confirmed by 31P NMR, mass-spectrometry and enzymatic hydrolysis.

Magnetic Resonance Spectroscopy↗

PubMed1999

RNA-binding and capping activities of proteins in rotavirus open cores.

Guanylyltransferases are members of the nucleotidyltransferase family and function in mRNA capping by transferring GMP to the phosphate end of nascent RNAs. Although numerous guanylyltransferases have been identified, studies which define the nature of the interaction between the capping enzymes of any origin and their RNA substrates have been limited. Here, we have characterized the RNA-binding activity of VP3, a minor protein component of the core of rotavirions that has been proposed to function as the viral guanylyltransferase and to direct the capping of the 11 transcripts synthesized from the segmented double-stranded RNA (dsRNA) genome of these viruses. Gel shift analysis performed with disrupted (open) virion-derived cores and virus-specific RNA probes showed that VP3 has affinity for single-stranded RNA (ssRNA) but not for dsRNA. While the ssRNA-binding activity of VP3 was found to be sequence independent, the protein does exhibit preferential affinity for uncapped over capped RNA. Like the RNA-binding activity, RNA capping assays performed with open cores indicates that the guanylyltransferase activity of VP3 is nonspecific and is able to cap RNAs initiating with a G or an A residue. These data establish that all three rotavirus core proteins, VP1, the RNA polymerase; VP2, the core capsid protein; and VP3, the guanylyltransferase, have affinity for RNA but that only in the case of the RNA polymerase is the affinity sequence specific.

Animals↗

PubMed2001

Synthesis and properties of mRNAs containing the novel "anti-reverse" cap analogs 7-methyl(3'-O-methyl)GpppG and 7-methyl (3'-deoxy)GpppG.

The ability to synthesize capped RNA transcripts in vitro using bacteriophage polymerases has been of considerable value in a variety of applications. However, Pasquinelli et al. [RNA (1995) 1:957-967] found that one-third to one-half of the caps are incorporated in the reverse orientation, that is, with the m7G moiety of m7GpppG linked by a 3'-5' phosphodiester bond to the first nucleotide residue of the RNA chain. Such reverse caps are unlikely to be recognized by eIF4E, based on previous studies, and thus complicate any comparison of the translational efficiencies of in vitro-synthesized mRNAs. We therefore designed two novel cap analogs, P(1)-3'-deoxy-7-methyguanosine-5' P3-guanosine-5' triphosphate and P(1)-3'-O,7-dimethylguanosine-5' P3-guanosine-5' triphosphate, that are, theoretically, incapable of being incorporated in the reverse orientation. The key reactions of pyrophosphate bond formation were achieved in anhydrous dimethylformamide solutions employing the catalytic properties of zinc salts. Structures were proven by 1H NMR. Transcripts produced with SP6 polymerase using "anti-reverse" cap analogs (ARCAs) were of the predicted length and indistinguishable in size and homogeneity from those produced with m7GpppG or GpppG. Analysis of the transcripts with RNase T2 and tobacco acid pyrophosphatase indicated that reverse caps were formed with m7GpppG but not with ARCAs. Both of the ARCAs inhibited cell-free translation with a K(I) similar to that of m7GpppG. Finally, the translational efficiency of ARCA-capped transcripts in a rabbit reticulocyte lysate was 2.3- to 2.6-fold higher than that of m7GpppG-capped transcripts. This suggests the presence of reverse caps in conventional in vitro-synthesized mRNAs reduces their translational efficiency.

Dinucleoside Phosphates↗

PubMed1994

Mutational analysis of yeast mRNA capping enzyme.

RNA guanylyltransferase (capping enzyme) catalyzes the transfer of GMP from GTP to the 5'-diphosphate end of mRNA. The capping reaction proceeds via an enzyme-guanylate intermediate in which GMP is linked covalently to a lysine residue of the enzyme. In the capping enzyme of Saccharomyces cerevisiae, GMP is attached to a 52-kDa polypeptide, identified as the product of the essential CEG1 gene. The amino acid sequence of the CEG1 protein includes a motif, Lys70-Thr-Asp-Gly, that is conserved at the active site of vaccinia virus RNA guanylyltransferase and which is similar to the KXDG sequence found at the active sites of RNA and DNA ligases. To evaluate the role of this motif in the function of the yeast enzyme, we have expressed the CEG1 protein in active form in Escherichia coli. Replacement of Lys70 or Gly73 with alanine abrogated enzyme-guanylate formation in vitro; in contrast, alanine substitutions at Thr71 or Asp72 merely reduced activity relative to wild-type enzyme. The K70A and G73A mutations were lethal to yeast, whereas yeast carrying the T71A and D72A alleles of CEG1 were viable. These results implicate Lys70 as the active site of yeast guanylyltransferase and provide evidence that cap formation per se is an essential function in eukaryotic cells.

Amino Acid Sequence↗