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Limiting concentrations of activated mononucleotides necessary for poly(C)-directed elongation of oligoguanylates

Selected imidazolide-activated nucleotides have been subjected to hydrolysis under conditions similar to those that favor their template-directed oligomerization. Rate constants of hydrolysis of the P-N bond in guanosine 5'-monophosphate 2-methylimidazolide (2-MeImpG) and in guanosine 5'-monophosphate imidazolide (ImpG), kh, have been determined in the presence/absence of magnesium ion as a function of temperature and polycytidylate [poly(C)] concentration. Using the rate constant of hydrolysis of 2-MeImpG and the rate constant of elongation, i.e., the reaction of an oligoguanylate with 2-MeImpG in the presence of poly(C) acting as template, the limiting concentration of 2-MeImpG necessary for oligonucleotide elongation to compete with hydrolysis can be calculated. The limiting concentration is defined as the initial concentration of monomer that results in its equal consumption by hydrolysis and by elongation. These limiting concentrations of 2-MeImpG are found to be 1.7 mM at 37 degrees C and 0.36 mM at 1 degrees C. Boundary conditions in the form of limiting concentration of activated nucleotide may be used to evaluate a prebiotic model for chemical synthesis of biopolymers. For instance, the limiting concentration of monomer can be used as a basis of comparison among catalytic, but nonenzymatic, RNA-type systems. We also determined the rate constant of dimerization of 2-MeImpG, k2 = 0.45 +/- 0.06 M-1 h-1 in the absence of poly(C), and 0.45 +/- 0.06 less than or equal to k2 less than or equal to 0.97 +/- 0.13 M-1 h-1 in its presence at 37 degrees C and pH 7.95.(ABSTRACT TRUNCATED AT 250 WORDS)

Limited proteolysis as a probe of the conformation and nucleic acid binding regions of nucleolin

Nucleolin, also called protein C23, is a RNA-associated protein implicated in the early stages of ribosome assembly. To study the general conformation and map the nucleic acid binding regions, rat nucleolin was subjected to limited proteolysis using trypsin and chymotrypsin in the presence or absence of poly(G). The cleavage sites were classified according to their locations in the three putative domains: the highly polar amino-terminal domain, the central nucleic acid binding domain, which contains four 90-residue repeats, and the carboxyl-terminal domain, which is rich is glycine, dimethylarginine, and phenylalanine. The most labile sites were found in basic segments of the amino-terminal domain. This region was stabilized by Mg2+. At low enzyme concentrations, cleavage by trypsin or chymotrypsin in the amino-terminal domain was enhanced by poly(G). Trypsin produced a relatively stable 48-kDa fragment containing the central and carboxyl-terminal domains. The enhanced cleavage suggests that binding of nucleic acid by the central domain alters the conformation of the amino-terminal domain, exposing sites to proteolytic cleavage. At moderate enzyme concentrations, the 48-kDa fragment was protected by poly(G) against tryptic digestion. At the highest enzyme concentrations, both enzymes cleaved near the boundaries between repeats 2, 3, and 4 with some sites protected by poly(G), suggesting that the repeats themselves form compact units. The carboxyl-terminal domain was resistant to trypsin but was cleaved by chymotrypsin either in the presence or in the absence of poly(G), indicating exposure of some phenylalanines in this region. These studies provide a general picture of the topology of nucleolin and suggest that the nucleic acid binding region communicates with the amino-terminal domain.

Ribosomal proteins S7 and L1 are located close to the decoding site ofE.coliribosome — affinity labeling studies with modified tRNAs carrying photoreactive probes attached adjacent to the 3′-end of the anticodon

Two photoreactive azidonitrophenyl probes have been attached to Yeast methionine elongator tRNA by chemical modification of the N6-(threoninocarbonyl)adenosine located next to the 3'-end of the anticodon. The maximum distance between the purine ring and the azido group estimated for the two probes is 16-17 and 23-24A, respectively. Binding and cross-linking of the uncharged, modified tRNAs to E. coli ribosomes have been studied with and without poly(A,U,G) as a message, under conditions directing uncharged tRNAs preferentially to the P-site. The modified tRNAs retain their binding activity and upon irradiation bind covalently to the ribosome with very high yields. Protein S7 is the major cross-linking target for both modified tRNAs, in the presence or absence of poly(A,U,G). Protein L1 and to a lesser extent proteins L33 and L27 have been found to be cross-linked with the short probe. Cross-linking to 168 rRNA reaches significant levels only in the absence of the message.

Oligomerization of deoxynucleoside-bisphosphate dimers: template and linkage specificity.

Evidence is presented that a poly(U) template selectively favors the oligomerization of the activated, 3'-5' pyrophosphate-linked dimer pdAppdAp, in comparison with the 3'-3' and 5'-5' linked dimers. In the absence of poly(U), the 5'-5' linked dimer is the most reactive, and chains are formed which are more than 60 monomer units in length.

The ATP binding site on rho protein. Affinity labeling of Lys181 by pyridoxal 5′-diphospho-5′-adenosine.

We have labeled the nucleoside triphosphate-binding domain of Escherichia coli rho factor with the ATP affinity analog [3H]pyridoxal 5'-diphospho-5'-adenosine (PLP-AMP). PLP-AMP completely inactivates the RNA-dependent ATPase activity of rho upon incorporation of 3 mol of reagent/mol of hexameric rho protein. Although the potency of PLP-AMP is enhanced when an RNA substrate such as poly(C) is present, the stoichiometry for inhibition remains the same as in the absence of poly(C). The nucleotide substrate ATP competes very effectively for the binding site and protects against PLP-AMP inactivation. A domain of rho called N2, which comprises the distal two-thirds of the molecule (residues 152-419) and encompasses the region proposed to bind ATP, is labeled specifically in the presence of poly(C). Amino acid sequence analysis of the single [3H]PLP-AMP labeled proteolytic fragment showed Lys181 to be the site of modification, suggesting that this residue normally interacts with the gamma-phosphoryl of bound ATP. These results agree with our proposed tertiary structure for the ATP-binding domain of rho that places this lysine residue in a flexible loop above a hydrophobic nucleotide-binding pocket comprised of several parallel beta-strands, similar to adenylate kinase, F1-ATPase, and related ATP-binding proteins. Parallel studies of rho structure and function by site-directed mutagenesis and chemical modification support this interpretation.

Peptide elongation factor 1 from yeasts: purification and biochemical characterization of peptide elongation factors 1 alpha and 1 beta (gamma) from Saccharomyces carlsbergensis and Schizosaccharomyces pombe.

Cytoplasmic elongation factor 1 alpha (EF-1 alpha) [corrected] was purified to homogeneity in high yield from the two different yeasts Saccharomyces carlsbergensis (S. carls.) and Schizosaccharomyces pombe (S. pombe). The purification was easily achieved by CM-Sephadex column chromatography of the breakthrough fractions from DEAE-Sephadex chromatography of cell-free extracts. The basic proteins have a molecular weight of 47,000 for the S. carls. factor and of 49,000 for the S. pombe factor. While the purified yeast EF-1 alpha s function analogously to other eukaryotic factors and the E. coli EF-Tu in Phe-tRNA binding and polyphenylalanine synthesis, the yeast factor unusually hydrolyzed GTP on yeast ribosomes upon addition of Phe-tRNA in the absence of poly(U) as mRNA. This novelty is probably owing to the yeast ribosomes, which are assumed to lack elongation factor 3-equivalent component(s). Trypsin and chymotrypsin selectively cleaved the two yeast factors to generate resistant fragments with the same molecular weight of 43,000 (by trypsin) and of 44,000 (by chymotrypsin), respectively. Those cleavage sites were characteristically protected by the presence of several ligands bound to EF-1 alpha such as GDP, GTP, and aminoacyl-tRNA. Based on the sequence analysis of the fragments generated by the two proteases, the partial amino acid sequence of the S. carls. EF-1 alpha was deduced to be in accordance with the N-terminal region covering positions (1) to 94 and two Lys residues at the C-terminal end of the predicted total sequence of the Saccharomyces cerevisiae (S. cerev.) factor derived from DNA analysis, except for a few N-terminal residues, confirming the predicted S. cerev. sequence at the protein level. EF-1 beta and EF-1 beta gamma were isolated and highly purified as biologically active entities from the two yeasts. EF-1 beta s from the two yeasts have the same molecular weight of 27,000, whereas component gamma of the S. carls. EF-1 beta gamma showed a higher molecular weight (47,000) than that of the S. pombe factor (40,000). It was also shown that a stoichiometric complex was formed between EF-1 alpha and EF-1 beta gamma from S. pombe. Furthermore, a considerable amount of Phe-tRNA binding activity was distributed in the EF-1H (probably EF-1 alpha beta gamma) fraction from freshly prepared cell-free extracts of yeast.

Interaction of polylysine with bovine factor Xa: Effect of divalent cations

Poly- L -lysine has been demonstrated to partially replace biological cofactors in the activation of prothrombin by factor Xa. The present study was initiated to determine if poly- L -lysine has an effect on the enzymatic activity of factor Xa in the absence of prothrombin. At low ionic strength(50 mM Tris-C1, pH 8.0, ambient temperature), poly- L -lysine inhibits amidase activity(S-2222) of bovine factor Xa with high affinity(Ki = 7 nM). The inhibition was readily reversed by 100 mM NaCl 2. The inhibition was also markedly reduced by the addition of 1.0 mM CaCl 2 but not by MnCl 2 or MgCl 2. All three metal ions enhance amidase activity in the absence of poly- L -lysine. Poly- L -lysine also inhibits the amidase activity of factor Xa from which the gamma-carboxyglutamic acid domain has been removed by limited proteolysis with chymotrypsin(factor Xa-GD) but with somewhat lower avidity(Ki = 35 nM). As with native factor Xa, calcium ions reduce the observed inhibition while either manganese or magnesium ions are much less effective. The amidase activity of factor Xa-GD is enhanced with any one of the three divalent cations. These results provide additional support for the existence of a functionally significant binding site for calcium ions outside of the gamma-carboxyglutamic domain of factor Xa.

Intracellular neutral carrier-based Ca2+ microelectrode with subnanomolar detection limit.

In intracellular electrolyte solutions a Ca2+-selective microelectrode based on the synthetic electrically neutral carrier N,N,N',N'-tetracyclohexyl-3-oxapentanediamide (ETH 129) shows an improved detection limit when compared with the so far widely used Ca2+ microelectrodes based on the neutral carrier ETH 1001. Detection limits are found at pCa = 9.2 in Ca2+ buffers containing an intracellular background of K+ (125 mM). Selectivity studies in mixed solutions show a preference of Ca2+ over Na+ of 6 X 10(5), over K+ of 1.6 X 10(6), and over Mg2+ of 5 X 10(6). The microelectrode does not suffer from significant interference by inorganic and organic inhibitors and by lipophilic cations and anions. The low detection limit is unchanged at least during the first eight hours of continuous contact with Ca2+ solutions. The EMF drift during the first hour of use is between 5 and 10 mV and is then reduced to about 1 mV/h. The changes in EMF induced between solution of pCa = 7 and pCa = 8 are reproducible within 24.7 +/- 0.4 mV (SD, n = 8, about 3 h). These electrode characteristics were found for single-barrelled microelectrodes of one micrometer diameter front-filled with a PVC-containing membrane phase. In the absence of poly(vinyl chloride) in the membrane phase irregular EMF response curves were obtained throughout. Preliminary punctures of ferret ventricular muscle cells indicate that the Ca2+ electrode response is not disturbed by the contact of a cytosolic milieu.

Copolymerization of Chloromethylstyrene and Divinylbenzene in the Absence or Presence of Poly(Vinyl Chloride)

Abstract Studies of the copolymerization of chloromethylstyrene (CMS) and divinylbenzene (DVB) were done in the presence or absence of poly-(vinyl chloride) (PVC) in order to prepare anion-exchange membranes of excellent performance by the paste method. The copolymerization rate decreases when PVC is added and increases when the DVB quantity is increased in the presence of PVC. The copolymerization rate of the isomers of CMS and DVB increases in the following order: p-DVB > m-DVB > p-ethylvinylbenzene > p-CMS > m-CMS ≈ m-ethylvinylbenzene. This order is not affected by the presence of PVC. The copolymerization of CMS and DVB takes place partially in PVC particles.

The mechanism of action of GTP on Ca2+ efflux from rat liver microsomal vesicles.

1. Guanosine 5'-[gamma-thio]triphosphate (GTP[S]), if added before GTP, blocks both Ca2+ efflux promoted by GTP and the effect of GTP on enhancement of inositol 1,4,5-triphosphate (IP3)-promoted Ca2+ release from preloaded microsomal vesicles. If, however, GTP[S] is added after GTP, it does not reverse the Ca2+ efflux promoted by GTP, nor does it inhibit IP3-promoted Ca2+ release. 2. The effect of GTP in enhancing IP3-promoted Ca2+ release is maintained after washing the microsomal vesicles free of added GTP. After this treatment, enhancement of IP3-promoted Ca2+ efflux can be observed in the absence of poly(ethylene glycol). 3. Electron microscopy shows that during GTP treatment of microsomal vesicles there is rapid production of very large vesicular structures, apparently produced by fusion of smaller vesicles. 4. Light-scattering changes are detectable during the fusion process. 5. Both Ca2+ efflux promoted by GTP and the enhancement of IP3-promoted Ca2+ release seen in the presence of GTP can probably be attributed to GTP-dependent vesicle fusion.

Characterization of a low-relative-molecular-mass prolyl 4-hydroxylase from the green alga Chlamydomonas reinhardii.

Prolyl 4-hydroxylase was partially purified and characterized from the unicellular green alga, Chlamydomonas reinhardii. This enzyme differed from all the animal and plant prolyl 4-hydroxylases studied so far in that its Mr was only about 40,000 by gel filtration, being thus less than one-sixth of those determined for the vertebrate and higher-plant enzymes. The algal enzyme did not hydroxylate to any significant extent chick-embryo protocollagen or triple-helical (Pro-Pro-Gly)10, whereas a low hydroxylation rate was found with denatured (Pro-Pro-Gly)10. Poly(L-proline), which is an effective inhibitor of the vertebrate enzymes but acts as a substrate for some higher-plant enzymes, was a good substrate. In the absence of poly(L-proline) the enzyme catalysed an uncoupled decarboxylation of 2-oxoglutarate. Studies of the Km values for the co-substrates and cofactors and the specificity of the 2-oxoglutarate requirement, as well as inhibition studies with selected 2-oxoglutarate analogues, suggested that the catalytic site of the algal enzyme is similar to, but not identical with, those of the vertebrate enzymes. The existence of distinct similarities was further demonstrated by an inhibition of the algal enzyme activity with a monoclonal antibody to the beta-subunit of human prolyl 4-hydroxylase. The amount of prolyl 4-hydroxylase activity in the algal cells was not altered by signals which recognize the presence or absence of the cell wall, as determined in studies on experimental cell-wall regeneration and wall-less mutants.

Evidence that the three-site model for the ribosomal elongation cycle is also valid in the archaebacterium Halobacterium halobium

The properties of the ribosomal elongation cycle were analyzed in the extreme halophilic archaebacterium Halobacterium halobium under conditions optimal for poly(U)-dependent poly(Phe) synthesis (60 mM Mg2+, 6.4 M monovalent cations). For tRNA binding studies the standard nitrocellulose-filter technique could not be used, since due to the high salt concentration the nucleic acids were retained quantitatively on the filters. Instead, an ultracentrifugation technique was applied, which revealed in saturation experiments that the following numbers of tRNA molecules could be bound per poly(U) programmed ribosome: (a) three deacylated tRNAPhe molecules, or (b) two Phe-tRNAPhe molecules in the A and P sites, or (c) one AcPhe-tRNAPhe molecule in either the A or P site (exclusion principle). In the absence of poly(U), AcPhe-tRNAPhe and deacylated tRNAPhe bound exclusively to the P site, whereas a low but significant binding of Phe-tRNAPhe to the A site was observed. Functional analyses were performed with ribosomes carrying a deacylated [14C]tRNA in the P site and a peptidyl-tRNA in the A site. The results demonstrate that the translocation reaction is not accompanied by a release of deacylated tRNA, but that the deacylated [14C]tRNA is released in the subsequent round of elongation.

Proteins from rat liver cytosol which stimulate mRNA transport. Purification and interactions with the nuclear envelope mRNA translocation system.

Two polysome-associated proteins with particular affinities for poly(A) have been purified from rat liver. These proteins stimulate the efflux of mRNA from isolated nuclei in conditions under which such efflux closely stimulates mRNA transport in vivo, and they are therefore considered as mRNA-transport-stimulatory proteins. Their interaction with the mRNA-translocation system in isolated nuclear envelopes has been studied. The results are generally consistent with the most recently proposed kinetic model of mRNA translocation. One protein, P58, has not been described previously. It inhibits the protein kinase that down-regulates the NTPase, it enhances the NTPase activity in both the presence and the absence of poly(A) and it seems to increase poly(A) binding in unphosphorylated, but not in phosphorylated, envelopes. The other protein, P31, which probably corresponds to the 35,000-Mr factor described by Webb and his colleagues, enhances the binding of poly(A) to the mRNA-binding site in the envelope, thus stimulating the phosphoprotein phosphatase and, in consequence, the NTPase. The possible physiological significance of these two proteins is discussed.

Aqueous polymerization of vinyl monomers in the presence of surfactants, 3. System: Acrylamide/poly(butyl acrylate) latex

AbstractThe kinetics of the polymerization of acrylamide initiated by potassium peroxodisulfate in aqueous medium is studied in the presence of poly(butyl acrylate) latex. The rate of acrylamide polymerization increases with the concentration of the latex in the reaction system. The increase is especially pronounced at concentrations of solid latex higher than 100 g per dm3 of polymerization mixture. The activation energy of the acrylamide polymerization in the presence of poly(butyl acrylate) latex is 85,8 kJ · mol−1. This value is substantially higher than the value of 70,7 kJ · mol−1 found for the activation energy of the polymerization of acrylamide in the absence of poly(butyl acrylate) latex. A set of reactions is proposed and the equation describing the simultaneous homopolymerization of acrylamide and grafting of poly(butyl acrylate) latex with acrylamide is derived.

Interfacial electron transfer in colloidal TiO2 accelerated by surface adsorption and the electric double layer

Interfacial electron transfer from colloidal TiO2 to methyl viologen (MV2+) has been studied for a wide range of ionic strengths and pH values. As in previous work, the rate increases with increasing pH as a result of a combination of electrostatic and thermodynamic factors. The slopes of log k vs. pH plots range from 0.3 to 1.1 as the electrolyte concentration is decreased from 1.0 to 0.001 mol dm–3. Also the slope for a neutral viologen (PVS) was only 0.18. This shows that electrostatics are the dominant term. By a comparision with conventional electrode kinetics, the dielectric constant in the Helmholtz layer was found to be 63, which is close to the value of 78 for pure water. This situation applies when there is no evidence of ion adsorption, i.e. within 3 pH units of the PZZP and at salt concentrations pH 9) and for Na2SO4 at low and high pH. This can result in significant changes in the reaction rates. At pH 9 NaCl and MgCl2 reduced the rate constants by a factor of 280 and 800, respectively. At pH 3, 0.02 mol dm–3 Na2SO4 was sufficient to increase the rate by a factor of 120, whereas 0.1 mol dm–3 NaCl did not appear to adsorb on the colloid and only increased the rate by a factor of 9. The yield of MV˙+ can be used as a direct probe for surface-oxidation reactions in TiO2 colloids. The net yield of MV˙+ after a 25 ns laser flash depends on the efficiency with which the photogenerated holes can react with electron donors. This was studied in the absence of poly(vinyl alcohol) at high (pH 11) and low (pH 3) pH. In both cases the efficiency was in the order glycerol > ethylene glycol, formaldehyde ≫ CH3O[CH2]2OCH3, methanol, isopropanol and suggests that the polyalcohol functional group helps in the adsorption of the alcohol on the TiO2 particles. This may explain in part the success of PVA as a polymer support in work with TiO2 colloids.

Mapping labeled sites in Escherichia coli ribosomal RNA: distribution of methyl groups and identification of a photoaffinity-labeled RNA region putatively at the peptidyltransferase center.

We have developed a method for the rapid localization of sites of ribosomal RNA labeling to limited regions (approximately 200 bases). The method is based on the formation and polyacrylamide gel electrophoretic separation of hybrids between restriction fragments of rrnB DNA and isotopically labeled rRNA and the subsequent determination of radioactivity across the gel. Using [3H]adenine-labeled rRNA as a control sample, we optimized experimental conditions with respect to a number of variables, including rRNA:DNA stoichiometric ratio, temperature of the annealing step, and levels of nucleases. An important result is that different rRNA X DNA hybrid fragments are obtained in different yields. The method was then applied to analyses of C3H3-labeled rRNA, giving results in good accord with known and proposed sites of rRNA methylation, and of rRNA that has been photoaffinity-labeled with 5-azido-2-nitrobenzoyl-[3H]Phe-tRNAPhe, a probe directed toward the peptidyltransferase center. The latter study showed a single major site of RNA labeling, falling within bases 2445-2668 of 23S rRNA. The extent of labeling was shown to be dependent on light-induced formation of a reactive intermediate and to be decreased in the absence of poly(uridylic acid) or in the presence of puromycin. The location of this major site of labeling is consistent with recent results obtained with an analogous tRNA photoaffinity label [Barta, A., Steiner, G., Brosius, J., Noller, H. F., & Kuechler, E. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 3607-3611] and with related genetic and biochemical studies of antibiotic interaction with ribosomes suggesting that the peptidyltransferase center falls within region V (bases 2043-2625) of 23S rRNA.

Photoaffinity labeling of DNA-dependent RNA polymerase from Escherichia coli with 8-azidoadenosine 5'-triphosphate.

A photoaffinity analogue of adenosine 5'-triphosphate (ATP), 8-azidoadenosine 5'-triphosphate (8-N3ATP), has been used to elucidate the role of the various subunits involved in forming the active site of Escherichia coli DNA-dependent RNA polymerase. 8-N3ATP was found to be a competitive inhibitor of the enzyme with respect to the incorporation of ATP with Ki = 42 microM, while uridine 5'-triphosphate (UTP) incorporation was not affected. UV irradiation of the reaction mixture containing RNA polymerase and [gamma-32P]-8-N3ATP induced covalent incorporation of radioactive label into the enzyme. Analysis by gel filtration and nitrocellulose filter binding indicated specific binding. Subunit analysis by sodium dodecyl sulfate and sodium tetradecyl sulfate gel electrophoresis and autoradiography of the labeled enzyme showed that the major incorporation of radioactive label was in beta' and sigma, with minor incorporation in beta and alpha. The same pattern was observed in both the presence and absence of poly[d(A-T)] and poly[d(A-T)] plus ApU. Incorporation of radioactive label in all bands was significantly reduced by 100-150 microM ATP, while 100-200 microM UTP did not show a noticeable effect. Our results indicate major involvement of the beta' and sigma subunits in the active site of RNA polymerase. The observation of a small extent of labeling of the beta and alpha subunits, which was prevented by saturating levels of ATP, suggests that these subunits are in close proximity to the catalytic site.

Crosslinking of Escherichia coli 50S ribosomal subunits with chlorambucilyl oligoprolyl phenylalanyl-tRNA molecular rulers.

A series of P-site probes, chlorambucilyl-(Pro)n-Phe-tRNAPhe, were prepared and reacted with poly(U)-directed Escherichia coli MRE 600 ribosomes. Upon binding of the probes to ribosomes, 90% of the cpm bound were not released following subsequent interaction with puromycin. In the absence of poly(U) or in the presence of poly(C), binding was limited to the amount of cpm bound if ribosomes were incubated in the presence of puromycin before adding modified tRNA and poly(U). AcPhe-tRNAPhe was a competitive inhibitor of chlorambucilyl Phe-tRNAPhe. Binding to 50S subunits was strongly stimulated by poly(U), while binding to 30S subunits was not. Crosslinked 50S proteins were analyzed by two-dimensional gel electrophoresis. Crosslinking with molecular rulers containing zero prolines led to poly(U)-dependent labeling of L1 and L27. With rulers containing five prolines, L6, L25, L28, and the group L18,23,24 were labeled. Analysis of crosslinked ribosomal RNA on sucrose density gradients revealed almost no cpm in the 16S or 23S peaks, but only in the 5S peaks. This was observed with molecular rulers containing either zero or five proline residues.

Effect of ribosome binding and translocation on the anticodon of tRNAPhe as studied by wybutine fluorescence.

The complexes of N-AcPhe-tRNAPhe (or non-aminoacylated tRNAPhe) from yeast with 70S ribosomes from E. coli have been studied fluorimetrically utilizing wybutine, the fluorophore naturally occurring next to the 3' side of the anticodon, as a probe for conformational changes of the anticodon loop. The fluorescence parameters are very similar for tRNA bound to both ribosomal sites, thus excluding an appreciable conformational change of the anticodon loop upon translocation. The spectral change observed upon binding of tRNAPhe to the P site even in the absence of poly(U) is similar to the one brought about by binding of poly(U) alone to the tRNA. This effect may be due to a hydrophobic binding site of the anticodon loop or to a conformational change of the loop induced by binding interactions of various tRNA sites including the anticodon.

Nuclear RNA is spliced in the absence of poly(A) addition

Splicing of newly formed nuclear RNA transcripts has been demonstrated during adenovirus type 2 (Ad2) mRNA formation in HeLa cells in the presence of “cordycepin,” 3′ deoxyadenosine, a drug that stops poly(A) addition to nuclear RNA. Nuclear RNA prepared from Ad2-infected cells after a 30 min label time in the presence or absence of 3′ deoxyadenosine was hybridized to and eluted from Ad2 DNA sequences in the transcription units of region E1b and region E2. The nuclear RNA from the 3′ dA-treated cells did not contain poly(A) but did contain Ad2-specific molecules ∼200 to 250 bases shorter than the spliced mRNAs of the control infected cells. In addition, the ∼2 kb RNA from Ad2 region E2 was shown to have sequences that lie more than 3.5 kb apart on the DNA, suggesting that correct cutting and splicing of the primary transcript in the absence of poly(A) synthesis had occurred. Therefore, although poly(A) addition usually precedes splicing during mRNA formation, poly(A) is not required for splicing.