Presence Of Polyuridylic Acid Research Articles

Studies on the Removal of Endogenous Messenger Ribonucleic Acid Activity from Rat Liver Microsomes: EFFECT OF REMOVAL ON POLYURIDYLIC ACID-DIRECTED INCORPORATION OF PHENYLALANINE

Abstract Rat liver microsomes preincubated for 12 min with all the factors necessary for amino acid incorporation do not incorporate phenylalanine in the absence of polyuridylic acid. In the presence of polyuridylic acid, preincubated microsomes show a 3- to 4-fold greater polyuridylic acid-directed phenylalanine incorporation than nonpreincubated microsomes. By varying preincubation time, temperature, and magnesium ion, adenosine triphosphate, or guanosine triphosphate concentration, the amount of phenylalanine incorporated during preincubation and the phenylalanine-incorporating activity remaining after preincubation are altered. Under these various conditions the response of preincubated microsomes to polyuridylic acid is shown to be directly related to the amount of amino acid incorporated during preincubation and inversely related to the amino acid-incorporating activity remaining after preincubation. Preincubated microsomes are more sensitive to low levels of polyuridylic acid and require higher levels of polyuridylic acid to saturate their capacity to incorporate phenylalanine. Furthermore, preincubated microsomes from which endogenous messenger ribonucleic acid activity has been removed bind greater amounts of polyuridylic acid.

Chromatography of Escherichia coli Ribosomes on Diethylaminoethyl Cellulose

Abstract Biologically active ribosomes of Escherichia coli can be purified by chromatography on diethylaminoethyl cellulose. Such ribosomes (DEAE-ribosomes) contain about 30% less protein than ribosomes washed by repeated centrifugation. In spite of the loss in protein, the DEAE-ribosomes have the same sedimentation values as washed ribosomes, are active in polypeptide synthesis, and are able to bind phenylalanyl soluble ribonucleic acid in the presence of polyuridylic acid but without guanosine triphosphate or any component of the cell sap. The loss of protein from the DEAE-ribosomes is concomitant with a loss of about 80% of the latent ribosomal DNase but none of the latent ribosomal RNase. Removal of the protein does not alter the response of the DEAE-ribosomes to various inhibitors of protein synthesis, including streptomycin. The DEAE-ribosomes differ from the washed ribosomes in their response to pH, NH4Cl, and magnesium. The activity of the DEAE-ribosomes changes little if at all over the range of pH from 6.9 to 8.5 whereas the washed ribosomes exhibit a broad optimum between pH 7.8 and 8.2. High concentrations (0.12 m) of NH4Cl produce a parallel increase in the binding of phenylalanyl soluble RNA and polypeptide synthesis with the DEAE-ribosomes but not with the washed ribosomes. The optimal magnesium concentration for polypeptide synthesis is 0.012 m with the DEAE-ribosomes as compared to 0.016 m with the washed ribosomes. Electrophoretic patterns of the ribosomal protein obtained from the DEAE-ribosomes were largely the same as those obtained from the washed ribosomes except for the absence of one protein band. These differences indicate that all of the ribosomes chromatographed on DEAE-cellulose have lost protein. The electrophoretic patterns of the ribosomal protein obtained from DEAE-ribosomes and washed ribosomes of E. coli K-12 was compared to ribosomal protein from similar ribosomal preparations of E. coli B.

Studies on the Formation of Transfer Ribonucleic Acid-Ribosome Complexes: I. THE EFFECT OF STREPTOMYCIN AND RIBOSOMAL DISSOCIATION ON 14C-AMINOACYL TRANSFER RIBONUCLEIC ACID BINDING TO RIBOSOMES

Abstract Streptomycin-sensitive 70S ribosomes which are dissociated into 50S and 30S particles by dialysis against 1 x 10-4 m magnesium acetate, and then reassociated in 0.02 m magnesium acetate, differ from nondissociated 70S ribosomes in 14C-aminoacyl soluble ribonucleic acid (sRNA) binding. 1. Reassociated ribosomes bind about twice as much 14C-phenylalanyl-sRNA in the presence of polyuridylic acid as nondissociated ribosomes. 2. 14C-Phenylalanyl-sRNA binding to reassociated ribosomes in the presence of polyuridylic acid is reduced 50% by streptomycin; this binding to nondissociated ribosomes is only slightly affected by streptomycin. 3. Streptomycin can inhibit 14C-isoleucyl-sRNA binding to reassociated ribosomes in the presence of polyuridylic acid, but stimulates this binding to nondissociated ribosomes. These effects are greatly influenced by the sRNA-ribosome ratio. Shearing 70S ribosomes by passage through a micropipette also produces Reactions 2 and 3 above, but not Reaction 1. The results indicate that various treatments of the ribosomes (dissociation, shearing) and sRNA to ribosome ratios modify the effects of streptomycin on 14C-aminoacyl-sRNA binding to ribosomes. In addition, the data suggest that different mechanisms may be involved in the effects of streptomycin on inhibiting protein synthesis and on stimulating ambiguous codon recognitions.

The behaviour of acetylphenylalanyl soluble ribonucleic acid in polyphenylalanine synthesis

In the presence of polyuridylic acid, the ribosomal system of Escherichia coli incorporates acetylphenylalanine from acetylphenylalanyl-sRNA into the N-terminal position during polyphenylalanine synthesis. In this case, acetylphenylalanyl-sRNA is also partially used as donor of phenylalanine in internal positions during polypeptide-chain formation. This peculiar incorporation of phenylalanine apparently occurs by a de-acetylation of acetylphenylalanyl-sRNA concomitant with peptidebond formation, since there is no detectable de-acetylation of free acetylphenylalanyl-sRNA. In the presence of E. coli endogenous mRNA instead of polyuridylic acid, acetylphenylalanyl-sRNA, surprisingly, is not used for protein synthesis.

Protein synthesis by free and bound rat liver ribosomes in vivo and in vitro

Sucrose density gradient centrifugation has been used to separate free ribosomes in the microsomal fraction of rat-liver cytoplasm from those bound to the lipoprotein membrane (endoplasmic reticulum). During in vivo labeling experiments, radioactive arginine becomes rapidly associated with membrane-bound ribosomes but not with free ribosomes. Further, in an in vitro protein synthesizing system membrane-bound ribosomes incorporate labeled phenylalanine while free ribosomes are virtually inert. These observations suggest that membrane-bound ribosomes are the major site of protein synthesis. The inactivity of the free ribosomes is presumably attributable to the absence of messenger RNA, for they readily incorporate phenylalanine in the presence of polyuridylic acid.

The synthesis of ribonucleic acid in sea urchin embryos

The sea urchin egg rapidly acquires the ability to synthesize protein after fertilization. This activation is due to a changing property of the ribosomes (1). Previous work has shown unfertilized egg ribosomes are capable of directed protein synthesis in the presence of polyuridylic acid (2, 3). The purpose of this report is to present evidence suggesting the hypothesis that the activation of ribosomes is a consequence of the activation of messenger RNA synthesis. Experiments will be described showing that the sedimentation properties of ribosomes from fertilized and unfertilized eggs are virtually identical. RNA synthesis is activated following fertilization, and a portion of this RNA is found in the ribosome fraction. The bulk of the newly synthesized RNA does not possess sedimentation properties of ribosomal RNA.

Reticulocyte protein synthesis: response of ribosome fractions to polyuridylic acid.

Reticulocyte ribosomes with sedimentation coefficients greater than 100S ("heavy" ribosomes) appear to be considerably more active in hemoglobin synthesis than are 78S ribosomes. When assayed for the ability to synthesize polyphenylalanine in the presence of polyuridylic acid, the 78S ribosomes and "heavy" ribosomes have similar activities. Polyuridylic acid inhibits incorporation by "heavy" ribosomes of amino acids other than phenylalanine.