Monoribosomal attachment to messenger ribonucleic acid in sodium fluoride-treated rabbit reticulocytes (Short Communication)

Mild proteolysis with Pronase selectively dissociates ribosomes not attached to mRNA into subunits; ribosomes attached to mRNA remain intact. A portion of monoribosomes from reticulocytes incubated with NaF resisted proteolytic dissociation. Recovery of mRNA from monoribosomes of NaF-treated reticulocytes therefore may be explained by persistent attachment of some monoribosomes to mRNA.     

Neurospora crassa glutamine synthetase. Translation of specific messenger ribonucleic acid in a cell-free system derived from rabbit reticulocytes.

The total reticulocyte lysate cell-free protein-synthesizing system was incubated in the presence of Neurospora crassa RNA. With the aid of an antibody directed against purified N. crassa glutamine synthetase, the synthesis of a specific protein was detected. This protein precipitates with antiglutamine synthetase using both direct and indirect procedures, migrates with the same molecular weight as the monomer of N. crassa glutamine synthetase when subjected to acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, and chromatographs as N. crassa glutamine synthetase on anthranilate-bound Sepharose. These data indicate the translation of the mRNA that codes for N. crassa glutamine synthetase. This RNA behaves as poly(A)-containing material when fractionated on oly(U)-Sepha-rose.     

Free cytoplasmic messenger ribonucleoprotein complexes from rabbit reticulocytes

Free cytoplasmic globin mRNA containing mRNP-particles were isolated from rabbit reticulocytes by zonal sucrose gradient centrifugation and their properties were compared with mRNP particles isolated in the same way from EDTA-dissociated reticulocyte polyribosomes. The average poly(A)-length of 9S mRNA from free cytoplasmic mRNP was 17–20 nucleotides being about two times shorter than the average poly(A)-length of polysomal 9S mRNA. The protein composition of the free cytoplasmic mRNP particles disclosed the absence of the 76,000 dalton protein which is associated with the 3poly(A)-segment of polysomal globin mRNA. It was concluded that free cytoplasmic mRNP-particles from rabbit reticulocytes can be classified as old mRNP in a post-translational phase. Free cytoplasmic mRNPs were translated in heterologous cell-free systems as well as in Xenopus laevis oocytes. Addition of hemin stimulated the synthesis of -globin in all systems, while the presence of the cap analogue m⁷G(5)p inhibited translation of free cytoplasmic mRNA completely. The latter finding suggested that free cytoplasmic mRNA has a 5 terminal cap. Shortening of the poly(A)-segment with concomitant loss of the 76,000 dalton protein may lead to less efficient translation of free cytoplasmic mRNP.     

Interaction between polyuridylic acid and rabbit globin messenger ribonucleic acid

Poly(U) binds to globin mRNA in 0.1m-NaCl. Studies with ribonuclease digestion of this complex suggest that there are polyadenylate-rich sequences in the mRNA containing about 30-40 adenylate residues. The sequences appear to be homogenous and of approximately the same length for both alpha- and beta-globin mRNA. They are most likely located at the 3' terminus of the molecule.     

A specific dimerization of rabbit beta-globin messenger ribonucleic acid.

Rabbit globin mRNA, when layered in low salt on 0.1 M-NaCl/sucrose gradients, separates into two peaks of material. Translation of these two RNA fractions in the wheat-germ cell-free system, hybridization against globin complementary DNA (cDNA) and cross-hybridization against cDNA species prepared from each fraction show that the first peak sedimenting at 10S is a alpha-globin mRNA and the second peak, sedimenting at approx. 15S, is beta-globin mRNA. The sedimentation rate of the beta-globin mRNA is concentration-dependent. By changing concentration and pH, it is indicated that in low-salt beta-globin mRNA adopts a conformation that leads to specific, but weak, self-dimerization during centrifugation in 0.1M-NaCl. This property permits rapid preparation of intact and relatively pure alpha- and beta-globin mRNA species.     

Studies on the secondary structure of ribosomal ribonucleic acid components of rabbit reticulocytes

The conformation in solution of fractionated 30 S and 19 S ribosomal RNA from rabbit reticulocytes has been studied by optical rotatory dispersion, analysis of thermal melting profiles and their derivatives, and spectrophotometric acid-base titration. From a consideration of the limitations of these methods, it has been possible to set limiting values on the degree of base-pairing and the lengths of the double helices: between 60 and 80% of the bases in 19 S and 30 S RNA are estimated to be paired. The paired segments are not shorter than 4 base pairs, and evidence from other sources is available which indicates that they are not longer than 8–16 base pairs. The spread of helix lengths is greater in the 30 S than in 19 S RNA; and other differences are noted. Several distinct populations of double helices, differing in their thermal stability, are present. Estimates are presented from spectrophotometric and titration data for the base compositions of the paired and unpaired regions.