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PubMed1976

Inhibition of initiation of protein synthesis by 7-methylguanosine-5'-monophosphate.

Translation of rabbit globin mRNA in a wheat germ protein-synthesizing system is inhibited by the nucleotide 7-methylguanosine-5'-monophosphate (m7G5'p) but not by other guanosine nucleotides without the 7-methyl group or with the phosphate in a different position. Translation of RNA of tobacco mosaic virus and poly(A) + HeLa RNA is also inhibited by m7G5'p. We show that m7G5'p prevents the association of mRNA with ribosomal subunits to form an initiation complex. We propose that m7G5'p interacts with a site on initiation factor(s) or ribosomes which is involved in mRNA recognition, presumably by binding to the 5'-terminal sequence m7G5'ppp. m7G5'p does not inhibit translation of poly(U) and RNA of satellite tobacco necrosis virus, which do not have the 5'-terminal sequence m7G5'ppp. In the case of RNA of satellite tobacco necrosis virus, some stimulation of its translation is consistently observed in the presence of m7G5'p; possible interpretations of this finding are discussed.

Cell-Free System↗
PubMed1993

Transcription initiation factor activity of vaccinia virus capping enzyme is independent of mRNA guanylylation.

Cytoplasmic extracts of vaccinia virus-infected HeLa cells blocked in DNA replication were capable of transcribing templates containing the minimal promoter sequences derived from three viral intermediate-stage genes (A1L, A2L, and G8R) but not promoters from early or late genes. One of three isolated components required for transcription copurified with the viral capping enzyme, a heterodimeric protein responsible for forming the 7-methyl-guanosine(5')triphospho(5')nucleoside [m7G(5')ppp(5')N-] structure at the 5' end of mRNAs, as had been reported using a template with another intermediate promoter [Vos, J. C., Sasker, M. & Stunnenberg, H. G. (1991) EMBO J. 10, 2553-2558]. Transcription factor activity was associated with partially, purified capping enzyme from infected cell extracts, homogeneous enzyme from purified virions, and recombinant viral enzyme from Escherichia coli. By transcribing truncated templates of different sizes, we determined that RNA chains of 35 nt were capped whereas those of 15 nt were not. Nevertheless, the capping enzyme was required for formation of short uncapped transcripts, indicating that capping and transcription initiation factor activities are independent functions.

Base Sequence↗
PubMed1995

Interaction of wheat germ protein synthesis initiation factor eIF-(iso)4F and its subunits p28 and p86 with m7GTP and mRNA analogues.

The binding of p28, p86, and native wheat germ eIF-(iso)4F with m7GTP and oligonucleotides was measured and compared. The purified subunits (p28, 28 kDa and p86, 86 kDa) of wheat germ protein synthesis initiation factor eIF-(iso)4F have been obtained from Escherichia coli expression of the cloned DNA (van Heerden, A., and Browning, K. S. (1994) J. Biol. Chem. 269, 17454-17457). The binding of the 5'-terminal cap analogue m7GTP to the small subunit (p28) of eIF-(iso)4F as a function of pH, temperature, and ionic strength is described. The mode of binding of p28 to cap analogues is very similar to the intact protein. Assuming that all tryptophan residues contribute to p28 and eIF-(iso)4F fluorescence, iodide quenching shows that all 9 tryptophan residues in p28 are solvent-accessible, while only 6 out of 16 tryptophan residues are solvent-accessible on the intact eIF-(iso)4F. The fluorescence stopped-flow studies of eIF-(iso)4F and p28 with cap show a concentration-independent conformational change. The rate of this conformational change was approximately 10-fold faster for the isolated p28 compared with the native eIF-(iso)4F. From these studies it appears that cap recognition resides in the p28 subunit. However, p86 enhances the interaction with capped oligonucleotides and probably is involved in protein-protein interactions as well. Both subunits are required for helicase activity.

Base Sequence↗
PubMed2004

A relaxed discrimination of 2'-O-methyl-GTP relative to GTP between de novo and Elongative RNA synthesis by the hepatitis C RNA-dependent RNA polymerase NS5B.

Several nucleotide analogues have been described as inhibitors of NS5B, the essential viral RNA-dependent RNA polymerase of hepatitis C virus. However, their precise mode of action remains poorly defined at the molecular level, much like the different steps of de novo initiation of viral RNA synthesis. Here, we show that before elongation, de novo RNA synthesis is made of at least two distinct kinetic phases, the creation of the first phosphodiester bond being the most efficient nucleotide incorporation event. We have studied 2'-O-methyl-GTP as an inhibitor of NS5B-directed RNA synthesis. As a nucleotide competitor of GTP in RNA synthesis, 2'-O-methyl-GTP is able to act as a chain terminator and inhibit RNA synthesis. Relative to GTP, we find that this analogue is strongly discriminated against at the initiation step ( approximately 150-fold) compared with approximately 2-fold at the elongation step. Interestingly, discrimination of the 2'-O-methyl-GTP at initiation is suppressed in a variant NS5B deleted in a subdomain critical for initiation (the "flap," encompassing amino acids 443-454), but not in P495L NS5B, which shows a selective alteration of transition from initiation to elongation. Our results demonstrate that the conformational change occurring between initiation and elongation is dependent on the allosteric GTP-binding site and relaxes nucleotide selectivity. RNA elongation may represent the most probable target of 2'-modified nucleotide analogues, because it is more permissive to inhibition than initiation.

Allosteric Site↗
PubMed1992

Microinjected U snRNAs are imported to oocyte nuclei via the nuclear pore complex by three distinguishable targeting pathways.

The inhibitory effects of wheat germ agglutinin and mAb 414 on the nuclear import of all types of U snRNAs indicate that they cross the nuclear envelope through the nuclear pore complex. However, the import of different U snRNAs occurs by kinetically distinct targeting pathways that can be distinguished from one another by the competitive effects of free trimethylguanosine cap dinucleotide (m3GpppG) and P(Lys)-BSA, an efficient synthetic karyophile based on the nuclear localization signal of SV40 large T antigen. The import of U snRNAs that contain 5' m3GpppN caps and are complexed by Sm proteins (U1, U2, U4, and U5) is competed by coinjection with free m3GpppG, indicating a shared transport factor, but not by P(Lys)-BSA. The import of U6 snRNA, which lacks a m3GpppN cap and is not complexed by the Sm proteins, is competed by P(Lys)-BSA but not by free m3GpppG. Thus, by the criterion of kinetic competition, U6 snRNA import is identical to that of the karyophilic proteins P(Lys)-BSA and nucleoplasmin. Uniquely, the import of U3 snRNA, which contains a m3GpppN cap but does not bind Sm proteins is not competed by either free m3GpppG or P(Lys)-BSA. Thus, U3 snRNA appears to be imported by a novel third kinetic pathway.

Amino Acid Sequence↗
PubMed1992

Characterization of antibodies against methyl-pppN cap structure: plant U3 small nucleolar RNA is recognized by these antibodies.

In eukaryotes, many small nuclear RNAs contain either a trimethylguanosine cap structure of a gamma-monomethyl (me) cap structure. Previously, we reported the characterization of anti-mepppG antibodies which recognize methyl-capped RNAs with G as the initiation nucleotide. We report here the preparation of antibodies against mepppN cap structure. Anti-mepppN antibodies recognized only mepppN from a mixture of mepppN and pppN and immunoprecipitated mepppA-capped U3 small nucleolar RNA from a mixture of cowpea cell RNAs. These anti-mepppN antibodies recognized methylated nucleoside triphosphates (mepppA, mepppC, mepppG and mepppU) with nearly equal efficiency; however, these antibodies did not recognize methyl phosphate or methylated mononucleotides. These antibodies will be useful in the identification and characterization of all methyl-capped RNAs no matter which is the initiation nucleotide.

Antibodies, Antinuclear↗
PubMed1997

Identification of a protein linked to the genomic and subgenomic mRNAs of feline calicivirus and its role in translation.

125I protein labelling of oligo(dT)-selected RNA from feline calicivirus (FCV)-infected cells revealed that the genomic and 2.4 kb subgenomic RNAs of FCV are linked to a 15 kDa protein (VPg). Proteinase K treatment of FCV RNA, to remove VPg, led to a decrease in the translatability of the RNA, but there was no obvious change in the site of RNA initiation. Addition of the cap analogue 7-methylGTP to in vitro translations had no effect on the translation of FCV RNA, suggesting that FCV RNA is translated by a cap-independent mechanism. Further evidence that FCV RNA is translated by an unusual mechanism was obtained by translating FCV RNA in vitro at a range of K+ concentrations. FCV RNA was able to direct translation at K+ concentrations at which cellular RNA translation was inhibited.

Animals↗
PubMed1984

Nucleotide pyrophosphatase from potato tubers. Purification and properties.

Purification of potato tuber nucleotide pyrophosphatase (EC 3.6.1.9) has been modified to furnish a rapid and reproducible procedure yielding a preparation purified 1800-fold and homogeneous in sodium dodecyl sulphate/polyacrylamide gel electrophoresis. The Mr of the enzyme, from gel filtration or sucrose density gradient centrifugation, is 343000 or 346000 respectively; and SDS electrophoresis indicates an Mr for the subunit of 74000. Analytical isoelectrofocusing reveals a broad isoelectric range of pH 8.3-8.7. The enzyme is a glycoprotein. The purified enzyme exhibits the previously reported activities versus pyrophosphate linkages located at either the 5'-OH or 3'-OH of nucleosides, and phosphodiester linkages in: (a) aryl esters of nucleoside 3'- and 5'-phosphates, p-nitrophenylphosphate and orthophosphate, and (b) nucleoside cyclic 2',3'-phosphates. However, the relative rates of activity towards these substrates, and the corresponding V values, differ significantly. The enzyme exhibits additional novel activities, including ability to cleave dinucleoside polyphosphates such as A(5')p2(5')A-A(5')p5(5')A, and aryl phosphonates. Contrary to previous reports, there is no activity towards nucleoside cyclic 3',5'-phosphates. The present preparation is also devoid of endonucleolytic activity, so that it specifically cleaves m7GMP from the 5'-terminal m7G(5')p3(5')Gm of intact reovirus mRNA. NAD+ was found to be the most effective inhibitor of enzyme activity versus thymidine 5'-p-nitrophenylphosphate, with a Ki = 0.1 mM. Kinetic analyses demonstrated competitive inhibition between these two substrates. Both 2',3'-cAMP and thymidine 3'-p-nitrophenylphosphate inhibit hydrolysis of NAD+ noncompetitively and vice-versa.

Chromatography, Thin Layer↗
PubMed1994

Influence of the four leader sequences of the human insulin-like-growth-factor-2 mRNAs on the expression of reporter genes.

The human insulin-like-growth-factor-2 (IGF-2) gene generates mRNAs with four different leader sequences, but with identical coding and trailing regions. Previous research has revealed that the leader-2-containing and leader-4-containing mRNAs are completely polysomal, whereas mRNAs possessing leader-3 are predominantly present in the untranslated free messenger ribonucleoprotein particle (mRNP), both in cell lines and in foetal liver tissue. To investigate the influence of the IGF-2 leader sequences on expression of the gene, IGF-2 leader-luciferase and leader-chloramphenicol acetyltransferase fusion constructs were transfected transiently into different cell lines. In these experiments, the levels of expression obtained by constructs with leader-1, leader-2 and leader-4 were very similar, both at the level of mRNA and protein. Leader-3, however, strongly repressed the expression of the fusion mRNA via an unknown mechanism. This repression appeared to be confined to nucleotides at positions 328-906 of the leader sequence. The remaining 5' part of the leader sequence was efficient both in RNA expression and in translation, but the 3' part of the leader (nucleotides 906-1180) again moderately repressed luciferase expression, possibly due to endonucleolytic cleavage in this region of the RNA. To evaluate the effect of the IGF-2 leaders on in vitro translation, leader-chloramphenicol acetyltransferase fusion mRNAs were synthesized and translated in reticulocyte lysates. Compared to a chloramphenicol acetyltransferase control RNA, leader-1-chloramphenicol acetyltransferase mRNA translated over 20-fold less efficiently, whereas leader-2 repressed translation of its chloramphenicol acetyltransferase mRNA moderately (3-5 fold). Despite a general improvement of the translation efficiency upon translation in HeLa lysate, these discrepancies with the transfection data persisted. Translation of leader-3-containing mRNAs in reticulocyte lysates was barely detectable. The whole 5' region of leader-3, up to nucleotide 614, could be shown to be repressive. Only leader-4 directed translation of the chloramphenicol acetyltransferase open reading frame efficiently. As with leader-1 and leader-2, this L4-chloramphenicol acetyltransferase mRNA translated in a cap-dependent manner under the conditions of our experiments; translation of this mRNA was relatively resistant to addition of cap analogue. We conclude that all four IGF-2 leader sequences differ in their translational properties. This makes it likely that changes in the translational machinery will affect the expression of the various IGF-2 mRNAs differentially.

Base Sequence↗
PubMed2006

The potyviral virus genome-linked protein VPg forms a ternary complex with the eukaryotic initiation factors eIF4E and eIF4G and reduces eIF4E affinity for a mRNA cap analogue.

The virus protein linked to the genome (VPg) of plant potyviruses is a 25-kDa protein covalently attached to the genomic RNA 5' end. It was previously reported that VPg binds specifically to eIF4E, the mRNAcap-binding protein of the eukaryotic translation initiation complex. We performed a spectroscopic study of the interactions between lettuce eIF4E and VPg from lettuce mosaic virus (LMV). The cap analogue m7GDP and VPg bind to eIF4E at two distinct sites with similar affinity (K(d) = 0.3 microm). A deeper examination of the interaction pathway showed that the binding of one ligand induces a decrease in the affinity for the other by a factor of 15. GST pull-down experiments from plant extracts revealed that VPg can specifically trap eIF4G, the central component of the complex required for the initiation of protein translation. Our data suggest that eIF4G recruitment by VPg is indirectly mediated through VPg-eIF4E association. The strength of interaction between eIF4E and pep4G, the eIF4E-binding domain on eIF4G, was increased significantly by VPg. Taken together these quantitative data show that VPg is an efficient modulator of eIF4E biochemical functions.

Binding Sites↗
PubMed1983

Control of synthesis of functional mRNA coding for phenylalanine ammonia-lyase from Rhodosporidium toruloides.

The regulation of functional mRNA coding for phenylalanine ammonia-lyase (PAL) from Rhodosporidium toruloides was investigated. Polyadenylic acid [poly(A)]-containing RNA was an efficient template for in vitro translation in rabbit reticulocyte lysate. Non-poly(A)-containing RNA did not stimulate in vitro protein synthesis. Several lines of experimental evidence indicate that mRNA from R. toruloides directs PAL synthesis in reticulocyte lysate: (i) the major radioactive product in immunoprecipitates when lysates, incubated with yeast poly(A)-containing RNA, were reacted with PAL-antiserum had the same molecular weight as native PAL (75,000); (ii) this major radioactive product competes with authentic PAL for binding to PAL-antiserum; and (iii) partial proteolytic peptide maps of the in vitro translation product were very similar to those of native PAL. The levels of functional mRNA coding for PAL, when R. toruloides was grown in different physiological conditions, were determined by quantitation of PAL synthesized in vitro when RNA was added to reticulocyte lysate. Functional PAL mRNA was six times higher in yeast grown on phenylalanine compared with glucose-phenylalanine minimal medium. No functional PAL mRNA was detected in yeast grown on glucose-ammonia minimal medium in the presence or absence of phenylalanine. These observed changes in functional PAL mRNA were similar to levels of PAL catalytic and antigenic activity. The kinetics of functional PAL mRNA synthesis and degradation were studied. Maximum levels of functional PAL mRNA were observed within 60 min of transfer to PAL-inducing growth conditions. Poly(A)-containing RNA and functional PAL mRNA were rapidly degraded when cells were transferred from phenylalanine to glucose-ammonia minimal medium, with half-lives of 25 and 10 min, respectively. Thus, it is suggested that the alterations in the amount of PAL in cells of R. toruloides grown in different physiological conditions primarily result from alteration in the amount of functional mRNA coding for the enzyme.

Ammonia-Lyases↗
PubMed1997

Improvement of the specific infectivity of the rubella virus (RUB) infectious clone: determinants of cytopathogenicity induced by RUB map to the nonstructural proteins.

A plasmid, Robo102, which contains a cDNA copy of the rubella virus (RUB) genomic RNA from which infectious transcripts can be synthesized in vitro, was recently developed (C. Y. Wang, G. Dominguez, and T. K. Frey, J. Virol. 68:3550-3557, 1994). To increase the specific infectivity of Robo102 transcripts (approximately 5 plaques/10 microg of transcripts), a modified reverse transcription-PCR method was used to amplify nearly 90% of the RUB genome in three fragments, which were then used to replace the corresponding fragments in Robo102. Replacement of a fragment covering nucleotides (nt) 5352 to 9759 of the RUB genome yielded a construct, Robo202, which produced highly infectious transcripts (10(4) plaques/microg), indicating the presence of an unrecognized deleterious mutation (or mutations) in this region of the Robo102 cDNA. Robo102 was based on the w-Therien strain of RUB, which forms opaque plaques in Vero cells, while the PCR replacement fragments were generated from a variant, f-Therien, which produces clear plaques in Vero cells. Although Robo202 contains over 4,000 nt from f-Therien, Robo202 virus produces opaque plaques. However, when the other two PCR fragments amplified from f-Therien (nt 1 to 1723 and nt 2800 to 5352) were introduced into Robo202, the resulting construct, Robo302, yielded transcripts that produced a virus that formed clear plaques. This indicates that the determinants of plaque morphology map to the regions of the genome covered by these two fragments, both of which are in the nonstructural open reading frame. Generation of Robo202/302 chimeras indicated that the most 5' terminal fragment (nt. 1 to 1723) had the greatest effect on plaque morphology. The plaque morphology was correlated with the ability of the viruses to kill infected cells. The only difference at the molecular level detected among the viruses was that the more cytopathic viruses produced more nonstructural proteins than did the less cytopathic viruses. This finding, as well as the mapping of the genetic determinants to the region of the genome encoding these proteins, indicates that the nonstructural proteins can mediate cell killing.

Animals↗
PubMed2001

Structural and thermodynamic behavior of eukaryotic initiation factor 4E in supramolecular formation with 4E-binding protein 1 and mRNA cap analogue, studied by spectroscopic methods.

The structural and thermodynamic behavior of the complex formation of eIF4E with either or both mRNA cap analogue (m7GTP, m7GpppA, or m7GpppG) and 4EBP1 has been investigated by spectroscopic measurements. Although the circular dichroism (CD) spectrum of eIF4E was little affected by the association with any cap analogue, the association constant of eIF4E with m7GpppA/G, estimated from the fluorescence quenching, was about 10 times larger than that with m7GTP. The van't Hoff analyses showed that the m7GpppA/G binding is enthalpy-driven with a large negative deltaH(o), and this is in contrast with the entropy-driven binding of m7GTP, where the positive deltaS(o) is large enough to overcome an increase of deltaH(o). This different behavior obviously originates in the interaction of the second nucleotide in m7GpppA with eIF4E, suggesting the importance of the nucleotide sequence linked to the m7Gppp terminal moiety, in addition to the specific interaction with the m7G base, for the recognition of mRNA cap structure by eIF4E. On the other hand, the CD spectra indicated that the binding of 4EBP1, an endogenous eIF4E-regulatory protein without having any defined secondary structure, shifted the m7GTP- or m7GpppA/G-bound eIF4E to an irregular structure, although such a structural change was not observed for eIF4E alone. The association constant of 4EBP1 with m7GTP- or m7GpppA/G-bound eIF4E was by two orders of magnitude larger than that with eIF4E alone. These results suggest the close interrelation in the supramolecular formation of 4EBP-eIF4E-mRNA cap structure.

Adaptor Proteins, Signal Transducing↗
PubMed1987

Regulation of mRNA entry into polysomes. Parameters affecting polysome size and the fraction of mRNA in polysomes.

The kinetics of labeled histone mRNA entry into polysomes was studied in nuclease-treated reticulocyte lysates. Added mRNA rapidly bound 1 or 2 ribosomes. However, the formation of full size polysomes required at least 16 min. The amount of mRNA bound to ribosomes reached a maximum (73%) within 2 min after mRNA addition and then declined slowly for the remainder of the experiment. Two initiation inhibitors, aurintricarboxylic acid and 7-methylguanosine 5'-triphosphate, were found to affect polysome size and the fraction of mRNA in polysomes in an opposite manner. These results suggest that initiation and reinitiation events may be intrinsically different. The relatively long time period required for the formation of large polysomes can be explained by large polysomes having higher initiation and/or reinitiation rates or slower elongation rates. These possibilities are not mutually exclusive. The results suggest that there exist several levels of control which can regulate polysome size and the fraction of mRNA in polysomes.

Animals↗
PubMed1979

Synthesis and translation of mRNA containing 5'-terminal 7-ethylguanosine cap.

Reovirus mRNA synthesis in vitro by the virion-associated RNA polymerase was only slightly (10 to 15%) diminished in the presence of 2 mM S-adenosylethionine. However, methyl group transfer from S-adenosylmethionine (0.05 mM) to the 5'-terminal cap structure, m7GpppGm in this mRNA was markedly inhibited (80%) under these conditions. Replacement of S-adenosylmethionine by S-adenosylethionine (5 mM) yielded mRNAs containing mainly (70%) 5'-terminal e7GpppGe and e7GpppG, but some of the products were unalkylated (5'-GpppG, ppG). The ethylated mRNAs, but not the unalkylated molecules, bound to wheat germ ribosomes and were translated essentially as well as the corresponding methylated mRNAs in wheat germ extracts and in nuclease-treated rabbit reticulocyte lysates. Protein synthesis directed by ethylated mRNAs in wheat germ extract was 80% decreased by 0.1 mM m7GMP. Under conditions of limited initiation, methylated mRNA bound to wheat germ ribosomes preferentially as compared to ethylated mRNA. The results document for the first time the synthesis of ethylated mRNA and support the hypothesis that N7-alkylation of the 5'-guanosine in caps, rather than methylation itself, is important for the enhancing effect of cap on the initiation of eukaryotic protein synthesis.

DNA-Directed RNA Polymerases↗