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W Filipowicz

Publications and source records attributed to W Filipowicz.

At least 91 records · Page 5Linked to original sources
PubMed1983

Initiation by RNA polymerase II and formation of runoff transcripts containing unblocked and unmethylated 5' termini.

Transcription of cloned adenovirus, beta-globin, and retrovirus long terminal repeat DNAs in HeLa whole-cell lysate was inhibited by S-adenosylhomocysteine. However, full-length 1.7-kilobase transcripts made on adenovirus 2 late promoter DNA contained 5'-terminal GpppA, consistent with specific initiation and runoff synthesis in the absence of product methylation. Formation of runoff transcripts including retrovirus RNAs that normally contain 5'-m7GpppGmpC was not decreased by replacing GTP with non-hydrolyzable analogs, and Rous-associated virus-2 runoff products made in the presence of GTP-gamma-S contained 5'-terminal gamma-S-pppGpC. The results indicate that capping and specific transcript synthesis by RNA polymerase II are not obligatorily linked in HeLa whole-cell lysate. Accurate initiation is dependent on ATP hydrolysis, and in contrast to GTP, replacement of ATP by 5'-adenylyl-imidodiphosphate blocked specific initiation of transcripts that start with either GTP (Rous-associated virus-2, Rous-associated virus-0) or ATP (beta-globin, adenovirus).

Adenoviruses, Human↗
PubMed1982

Circularization of linear viroid RNA via 2'-phosphomonoester, 3', 5'-phosphodiester bonds by a novel type of RNA ligase from wheat germ and Chlamydomonas.

A novel type of RNA ligase activity in extracts of wheat germ or Chlamydomonas requires 2', 3'-cyclic phosphate and 5'-phosphate ends for ligation to form a 2'-phosphomonoester, 3',5'-phosphodiester bond. Using 5'-3 2P-labeled linear PSTV, we demonstrate that RNase T1-nicked viroid predominantly forms (formula; see text) U-bonds. Natural linear PSTV, however, forms mainly (formula; see text) A-bonds upon enzymatic circularization. We show that natural linear PSTV RNA has nicks between C181 and A182, or between C348 and A349, and that consequently C181 and C348 carry 2',3'-cyclophosphate termini.

Chemical Phenomena↗
PubMed1982

RNA ligation via 2'-phosphomonoester, 3'5'-phosphodiester linkage: requirement of 2',3'-cyclic phosphate termini and involvement of a 5'-hydroxyl polynucleotide kinase.

Extracts of wheat germ contain a RNA ligase activity that catalyzes the conversion of linear polyribonucleotides into covalently closed circles. As reported previously, this enzyme joins two ends of a RNA substrate via a 2'-phosphomonoester, 3',5'-phosphodiester linkage. In the present work we provide evidence that a 2',3'-cyclic phosphate group at the 3' terminus is required for RNA ligation and that the 5'-hydroxyl end is phosphorylated before the two RNA ends are joined. We report on the presence of 5'-hydroxyl polynucleotide kinase and polynucleotide 2',3'-cyclic phosphate 3'-phosphodiesterase activities in wheat germ extracts. A possible involvement of these enzymes in the ligation process and a potential role of the newly described ligation pathway in RNA processing are discussed.

Cell-Free System↗
PubMed1981

Binding of ribosomes to linear and circular forms of the 5'-terminal leader fragment of tobacco-mosaic-virus RNA.

The sequence of the 5'-terminal leader fragment preceding the AUG codon in the RNA of tobacco mosaic virus (TMV), tomato strain, SPS isolate, has been determined. This RNA, similarly to the RNAs of the U1 and Dahlemense strains of TMV [Kukla et al. (1979) Eur. J. Biochem. 98, 61--66] has the 7-methylguanosine(5')triphospho(5')guanosine cap separated from the initiation codon by a long stretch of nucleotides devoid of guanosine residues. The RNase-T1-resistant 73-nucleotide-long leader fragment of TMV RNA from the SPS isolate was assayed for its ability to interact with eukaryotic and prokaryotic ribosomes. The linear fragment, labelled either at its 5' or 3' end, efficiently formed disome initiation complexes when incubated with wheat-germ protein-synthesis extract. In contrast to its linear counterpart, the circular covalently closed RNA leader fragment, obtained in a reaction catalysed by T4 RNA ligase, was unable to interact with wheat germ ribosomes. Both kinds of leader fragment bound equally well to Escherichia coli 70-S ribosomes. The results offer further support to the notion that in eukaryotic initiation the free 5' end (either capped or uncapped) is required for mRNA interaction with ribosomes. Furthermore, they suggest that both ribosomes found in disome initiation complexes with the TMV RNA leader fragment enter the mRNA sequentially via the free 5' terminus.

Base Sequence↗
PubMed1981

The cell-free protein synthesis system from the 'slime' mutant of Neurospora crassa. Preparation and characterisation of importance of 7-methylguanosine for translation of viral and cellular mRNAs.

A simple procedure for preparation of a cell-free protein synthesis system (23000 X g supernatant) from the protoplast-like 'slime' mutant of Neurospora crassa is described. A variety of messenger RNAs of viral and cellular origin could be efficiently and faithfully translated in this system into proteins with Mr as large as 180000. The importance of the 7-methylguanosine cap for mRNA translation in the Neurospora system was studied in detail using the cap analogs and chemically decapped messengers. As in the case of reticulocyte lysate or wheat germ extract, the extent of m7G requirement for mRNA translation in a fungal extract strongly depended on translation conditions such as incubation temperature or concentration of potassium ions, mRNA and 23000 X g supernatant protein.

Animals↗
PubMed1980

Suppression of induction of SOS functions in an Escherichia coli tif-1 mutant by plasmid R100.1.

The tif-1 mutation in the recA gene of Escherichia coli caused, at 40 degrees C, lethal cell filamentation, induction of the recA protein, mutagenesis, and, in lambda lysogens, prophage induction. The presence of plasmid R100.1 in tif-1 strains suppressed tif-mediated cell filamentation and killing, recA protein induction, and prophage induction in lysogens. It also reduced mutagenesis in a tif-1 sfiA11(R100.1) strain. Plasmids F'lac, P1, and pMB9, in contrast, had little or no effect on tif-mediated induction of lambda. The presence of R100.1 did not inhibit the induction of the recA protein or of lambda by ultraviolet irradiation or mitomycin C treatment of tif-1(R100.1) or tif-1(lambda)(R100.1) strains.

Adenine↗
PubMed1979

Binding of ribosomes to 5'-terminal leader sequences of eukaryotic messenger RNAs.

Tobacco mosaic virus and turnip yellow mosaic virus genome RNAs, which have long (greater than or equal to 69 nucleotides) 5'-terminal leader sequences preceding the AUG initiation codon, can form disome initiation complexes when incubated with a wheat germ extract and sparsomycin. In tobacco mosaic virus RNA, whereas one ribosome in such complexes presumably occupies the AUG-containing initiation site, the other is located within the RNA leader sequence. The results favor a model of eukaryotic initiation in which ribosomes first bind to the 5' end of the mRNA independently of AUG, then advance to the AUG initiation codon, where protein synthesis ensues.

Base Sequence↗
PubMed1978

5'-Terminal 7-methylguanosine and mRNA function. The effect of enzymatic decapping and of cap analogs on translation of tobacco-mosaic-virus RNA and globin mRNA in vitro.

1. Decapped tobacco mosaic virus (TMV) RNA and rabbit globin mRNA were prepared by enzymic treatment of RNAs with nucleotide pyrophosphatase purified from potato. The extent of removal of 5'-terminal 7-methylguanosine 5'-monophosphate (m7GMP) from TMV RNA was at least 97% as estimated by labeling of the 5' termini in vitro with S-adenosyl[methyl-3H]methionine catalysed by vaccinia virus methyltransferases. 2. The effect of enzymic decapping was compared with the effect of cap analogs on mRNAs translation in a nuclease-treated rabbit reticulocyte lysate and in a wheat germ extract. When translation was studied at low K+ concentration, little or no dependence on 5'-terminal 7-methylguanosine was found with either cell-free system. The importance of the 5'-terminal cap for the efficient translation of TMV RNA and globin mRNA increased as the concentration of K+ in a protein-synthesis system was raised. In a reticulocyte lysate analogs and enzymic decapping had a similar effect on translation. In a wheat germ extract, mRNA decapping resulted in a more pronounced decrease of mRNA activity, presumably due to the increased susceptibility of decapped mRNAs to the nucleases present in this protein synthesis system. 3. The requirement for a 5'-terminal cap was similar for the synthesis of 130,000-Mr and 165,000-Mr polypeptides coded by TMV RNA. This indicates that both proteins may be initiated at the common site close to the 5' terminus.

Animals↗
PubMed1977

Removal of 5'-terminal m7G from eukaryotic mRNAs by potato nucleotide pyrophosphatase and its effect on translation.

The procedure for isolation of nucleotide pyrophosphatase (E.C. 3.6.1.9.) from potato has been modified to yield an endonuclease-free preparation purified 2300-fold. The enzyme was used for specific cleavage of pyrophosphate linkages in the 5'-terminal cap (m7GpppN) of several eukaryotic messenger RNAs. Enzymatic removal of 5'-terminal pm7G from reovirus, rabbit globin and Artemia salina mRNAs resulted in an almost complete loss (greater than 80%) of their template activities in a cell-free protein synthesizing system from wheat germ. Incubation with nucleotide pyrophosphatase did not decrease the translation of phage f2 RNA in an Escherichia coli cell-free system.

Animals↗
PubMed1976

Polypeptide chain initiation in eukaryotes: initiation factor requirements for translation of natural messengers.

A protein-synthesizing system consisting of ribosomes and supernatant of undeveloped Artemia saline embryos is used for assay of mRNA translation and initiation factors. This system contains the components needed for chain elongation but has low levels of mRNA and initiation factors. Exogenous mRNA is readily translated upon addition of high-speed supernatant or ribosomal salt wash of developing embryos as a source of initiation factors (IF). This requirement can be largely satisfied by a mixture of the reticulocyte factors IF-MP, IF-M3, IF-M2A, and IF-M2B. A. salina IF-M1, which is present in undeveloped embryo supernatant, can be inactivated by A. salina IF-M1 antibody without affecting translation.

Animals↗
PubMed1976

A protein binding the methylated 5'-terminal sequence, m7GpppN, of eukaryotic messenger RNA.

Ribosomal salt washes of Artemia salina embryos contain a protein(s) that binds [3H]m7GpppGpC and [3H]m7GpppGmpC, as measured by retention on nitrocellulose membrane filters. These oligonucleotides correspond in structure to the methylated 5'-terminal sequences (caps) present in many eukaryotic mRNAs. The cap binding protein does not bind the unmethylated counterparts of caps, e.g., [32P]GpppGpCp, or a derivative of m7GpppGmpC containing ring-opened m7G. None of the purified initiation factors IF-MP, IF-M2A, IF-M2B, IF-M3, or IF-MI binds the m7G-containing oligonucleotides.

Base Sequence↗
PubMed1976

Activity of methoxyamine-modified f2 RNA in initiation and elongation steps of protein synthesis.

1. Messenger activity of phage f2 RNA modified with methoxyamine under non-denaturing conditions was studied in E. coli-free system. The incorporation of amino acids into phage polypeptides was decreased, and the synthesis of phage-specific proteins was diminished. The RNA replicase synthesis was more affected than synthesis of coat protein. The impaired messenger activity of the methoxyamine-modified f2 RNA was due to the blocking of elongation process by modified cytosines present in RNA chain. 2. Specificity of f2 RNA to stimulate ribosomal binding predominantly at the coat protein initiation site was not affected by methoxyamine-treatment, as demonstrated by unchanged binding of f[3H]Met-tRNA and [14C]alanyl-tRNA to ribosomes. 3. Unfolding of f2 RNA molecule on treatment with methoxyamine in the presence of guanidine-HCl resulted in a significant increase of RNA capacity to direct fMet-tRNA binding to ribosomes. Sucrose-density gradient profiles revealed the formation of polysome-like initiation complexes indicating that ribosomes were able to bind at many hitherto inaccessible initiation codons in RNA molecules. fMet-tRNA bound to ribosomes in the presence of unfolded RNA was found to be fully reactive with puromycin.

Cell-Free System↗