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R E Rhoads

Publications and source records attributed to R E Rhoads.

At least 109 records · Page 6Linked to original sources
PubMed1984

Purification of the messenger RNA cap-binding protein using a new affinity medium.

The p-aminophenyl gamma-ester of 7-methylguanosine 5'-triphosphate (m7GTP) was synthesized and coupled to Sepharose 4B. A 0.5 M salt extract of rabbit reticulocyte ribosomes was passed over a column containing the affinity medium. After extensive washing, a solution of m7GTP was passed through the column, and a single polypeptide species of 24 kilodaltons (kDa) was eluted. This had an electrophoretic mobility identical with that of the mRNA cap-binding protein. This assignment was confirmed by the fact that the eluted material was enriched nearly 200-fold in the ability to specifically bind 32P-labeled capped oligonucleotides. A control affinity medium consisting of GTP similarly coupled to Sepharose failed to retain the 24-kDa species. The postribosomal supernatant fraction yielded slightly more of the 24-kDa species than the ribosomal wash fraction when passed over this affinity medium.

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PubMed1983

Translational recognition of messenger ribonucleic acid caps as a function of pH.

The degree to which cell-free translation of eukaryotic mRNA is stimulated by the presence of a 5'-terminal 7-methylguanosine-containing cap is affected by a variety of factors including ionic strength, temperature, mRNA concentration, and the type of mRNA. In this report, we show that pH also affects cap dependence. Translation of globin mRNA from which the cap had been enzymatically removed was relatively insensitive to pH compared with capped mRNA. Also, at low pH (6.6-7.2), the cap analogue m7GTP caused little inhibition of globin mRNA translation in a cell-free system whereas at higher pH the degree of inhibition increased. Finally, the overall extent to which globin mRNA translation could be inhibited at saturating concentrations of m7GTP increased with increasing pH. It is also shown that the pKa of the N-1 proton of m7GTP is affected by mono- and divalent cations. At the K+ and Mg2+ concentrations optimal for cell-free translation, the pKa is approximately 7.4. Since the pH optimum for translation is near 7.6, both keto and enolate forms of m7G are present in appreciable amounts. One interpretation for the observed change in cap dependence with pH is that only the enolate form of m7G is recognized by the cap-binding protein.

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PubMed1983

Structure of the 5' terminus of hen oviduct lysozyme messenger ribonucleic acid.

Lysozyme mRNA (mRNAlys) was purified from hen oviduct poly(A)-containing RNA by hybridization, labeled with NaB[3H]4 and digested with RNase T1. This revealed the presence of equal amounts of two major oligonucleotides having structures of m7Gppp(Np)7 and m7Gppp(Np)4 plus minor amounts of m7Gppp(Np)2 and m7GpppNp. The total mRNAlys contained the cap structures m7Gpppm6Am, m7GpppGm, m7GpppAm, m7GpppCm, m7GpppA, and m7GpppG, in decreasing order of abundance. The m7Gppp(Np)7 oligonucleotide contained only A-caps and the m7Gppp(Np)4, only G-caps. 32P-labeled 5'-terminal T1-oligonucleotides were prepared, and at least 12 different types were observed, the most abundant being m7Gppp(Np)7 and m7Gppp(Np)4. Their sequences were determined to be m7Gppp(m6)AmNmUCCCG and m7GpppGmNmAG. Taken together with the findings of Grez et al. (Grez, M., Land, H., Giesecke, K., Schutz, G., Jung, A., and Sippel, A. E. (1981) Cell 25, 743-752), these results indicate that in the genomic sequence AGCTTGCAGTCCCGT, 52% of the mRNAlys molecules begin at the underlined A residue and 38% at the underlined G residue.

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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.

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PubMed1981

Heterogeneity of the 5' terminus of hen ovalbumin messenger ribonucleic acid.

The 5'-terminal sequence of hen ovalbumin mRNA was investigated using a novel labeling method. Ovalbumin mRNA was purified by hybridization to complementary DNA coupled to cellulose. The mRNA thus purified was shown to be 97.9% pure by hybridization with plasmid DNA containing sequences to the messengers coding for conalbumin and ovomucoid, the next two most abundant messengers of oviduct. After digestion with RNase T1 and alkaline phosphatase, 5'-terminal capped oligonucleotides were selected by binding to anti-m7G-Sepharose. These were then labeled using RNA ligase and [5'-32P]pCp, separated by two-dimensional gel electrophoresis, and sequenced by partial digestion with base-specific ribonucleases. A nested set of three capped oligonucleotides was identified. Their structures and relative abundances were m7GpppAUACAG, 3% m7GpppACAUACAG, 61+; and m7GpppGUACAUACAG, 36%.

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PubMed1978

Translational recognition of the 5'-terminal 7-methylguanosine of globin messenger RNA as a function of ionic strength.

The translation of rabbit globin mRNA in cell-free systems derived from either wheat germ or rabbit reticulocyte was studied in the presence of various analogues of the methylated 5' terminus (cap) as a function of ionic strength. Inhibition by these analogues was strongly enhanced by increasing concentrations of KCl, K(OAc), Na(OAc), or NH4(OAc). At appropriate concentrations of K(OAc), both cell-free systems were equally sensitive to inhibition by m7GTP. At 50 mM K(OAc), the reticulocyte system was not sensitive to m7GMP or m7GTP, but at higher concentrations up to 200 mM K(OAc), both nucleotides caused strong inhibition. The compound in m7G5'ppp5'Am was inhibitory at all concentrations of K(OAc) ranging from 50 to 200 mM, although more strongly so at the higher concentrations. Over the same range of nucleotide concentrations, the compounds GMP, GTP, and G5'ppp5'Am were not inhibitors. The mobility on sodium dodecyl sulfate-polyacrylamide electrophoresis of the translation product was that of globin at all K(OAc) concentrations in the presence of m7GTP. Globin mRNA from which the terminal m7GTP group had been removed by chemical treatment (periodate-cyclohexylamine-alkaline phosphatase) or enzymatic treatment (tobacco acid pyrophosphatase-alkaline phosphatase) was translated less efficiently than untreated globin mRNA at higher K(OAc) concentrations, but retained appreciable activity at low K(OAc) concentrations.

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PubMed1978

Chromatography of ovalbumin messenger ribonucleic acid on complementary deoxyribonucleic acid-cellulose.

DNA complementary to ovalbumin mRNA and covalently bound to cellulose (cDNA-cellulose) was synthesized using avian myeloblastosis virus RNA-directed DNA polymerase. High concentrations of actinomycin D (200 migrogram/ml) were required to produce 97% inhibition of double-stranded DNA synthesis, but mRNA transcription was only slightly inhibited (14%). The conditions used for binding of mRNA to cDNA-cellulose permitted complete hybridization of ovalbumin mRNA in 10 min while stable poly(A):(dT) hybrids failed to form. The temperature at which 50% of the ovalbumin mRNA activity was eluted from cDNA-cellulose was 62 degrees in 0.01 M Tris.HCl. When a batchwise procedure of hybridization and elution was used, the total recovery of ovalbumin mRNA activity applied to the cDNA-cellulose was greater than 98%, indicating little if any degradation of mRNA. Ovalbumin mRNA activity eluted in each chromatographic run was 50 to 70% of that originally used for the synthesis of the cDNA-cellulose. When total polysomal RNA was subjected to chromatography, the bound fraction consisted of ovalbumin mRNA, rRNA, and material behaving like fragments of ovalbumin mRNA. Applying this fraction to cDNA-cellulose a second time eliminated the rRNA but not the presumptive fragments. Ovalbumin mRNA purified either once or twice was enriched between 43- and 56-fold over polysomal RNA in translational activity.

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PubMed1975

Ovalbumin messenger ribonucleic acid. Purification and fractionation on the basis of polyadenylate content by thermal elution from oligodeoxythymidylate-cellulose.

Ovalbumin messenger RNA was purified from hen oviduct by immunoprecipitation of polysomes and oligo(dT)-cellulose chromatography. Two steps were introduced to improve the separation of mRNA and rRNA by oligo(dT)-cellulose. First, aggregates of mRNA and rRNA were dissociated by heating at 65degrees for 10 min before chromatography. Second, elution of the mRNA was achieved by stepwise increases in temperature rather than by lowering the ionic strength. Ovalbumin mRNA activity was eluted primarily in RNA fractions eluting between 45degrees and 55degrees. Polyacrylamide gel electrophoresis indicated that the ovalbumin mRNA thus obtained was essentiallyyy free of rRNA. The poly(A) content of various thermally eluted fractions was assayed by two methods. In the first (Favre, A., Bertazzoni, V., Berns, A.J.M., and Bloemendal, H. (1974) Biochem. Biophys. Res. Commun. 56, 273-280), the increase in fluorescence intensity of bound ethidium bromide was used to follow the formation of double-stranded RNA during titration of the mRNA with poly(U). In the second (Bishop, J. O., Rosbash, M., and Evans, D. (1974) J. Mol. Biol. 85, 75-86), poly(A) content was derived from the radioactivity remaining acid-insoluble after annealing mRNA fractions with [3H]poly(U) and treating with ribonuclease A. Both methods indicated that ovalbumin mRNA fractions eluting at higher temperatures contained greater amounts of poly(A). Values ranged from 44 to 248 mol of AMP/mol of mRNA, assuming 2200 total nucleotide residues for ovalbumin mRNA (Shapiro, D. J., and Schimke, R. T. (1975) J. Biol. Chem. 250, 1759-1764). Translational specific activities in a rabbit reticulocyte lysate system were essentiall constant for all fractions. From the binding of ethidium bromide it could be estimated that approximately 50% of the nucleotide residues in ovalbumin mRNA are base-paired.

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