Messenger Ribonucleoprotein and Initiation Factors in Rabbit‐Reticulocyte Polyribosomes

Abstract

EDTA dissociation of reticulocyte polyribosomes liberates globin mRNA as a nucleoprotein. An attempt to compare the proteins forming the mRNA * protein complexes with the initiation factors of protein synthesis is presented here.Two conditions are described under which initiation factors are removed from hemoglobinsynthesizing ribosomes. One is after treating the polyribosomes with a high salt concentration ; the other is by isolating ribosomes from reticulocytes at an advanced state of maturation which have lost their initiation factors. I n both cases it is shown that the mRNA remains associated with the ribosomes and with the proteins forming the mRNA * protein.Moreover both mRNA and mRNA -protein are translatable in a heterologous cell-free system and their translation is stimulated to the same extent by the addition of reticulocyte initiation factors.Initiation factors are therefore different physically and functionally from the proteins of the mRNA -protein. and little information has been provided regarding the biological significance of these mRNA * protein associations. Some progress towards understanding the functional role of mRNA-protein has been made in the rabbit reticulocyte system where the 15-S mRNA ' protein (in which form globin messenger RNA is released from the polyribosomes by EDTA treatment) can be isolated in a nearly homogeneous form. The proteins attached to globin mRNA are well defined and a t least one of them appears to be required for binding mRNA to native 40-5 ribosomal subunits washed with deoxycholate [4].It was therefore of interest to study the possible relationship between these mRNA * protein proteins and the factors isolated by concentrated-salt washing of ribosomes which have been shown to be required for initiation and to be specific for mRNA recogniAbbreviations. mRNA * protein, messenger ribonucleoprotein; Brij 35, polyoxyethylene cetylether.Definition. Asso unit, the quantity of material contained in 1 ml of a solution which has an absorbance of I at 260 nm, when measured in a 1-cm path-length cell.tion [6-81. However, a direct comparison has been impossible to perform since initiation factors have not yet been p d e d to homogeneity. I n order to compare these seemingly separate classes of proteins we have used two types of ribosomes which are both dependent on the addition of initiation factors in order to synthesize globin chains. These are ribosomes washed in 0.5 M KC1 [6,7] and ribosomes taken from a mature population of reticulocytes [Y].We have shown that in both cases the major part of the globin mRNA is still attached to the remaining population of monomers and subunits, and that their lack of activity is primarily due to the loss of their initiation factors. Moreover, the mRNA remains attached to the ribosomes in the form of a nucleoprotein complex apparently identical to the mRNA * protein isolated from normal polyribosomes. Removal of initiation factors does not therefore release the mRNA -protein complexes. Studies of the translation of mRNA and mRNA.prote...

The role of ribosomal RNA in protein synthesis

“…It is now obvious that the interplay between the ribosome and cytoplasmic as well as membrane components may significantly affect the protein synthetic capability of the ribosomal machinery. Factors required for the initiation, elongation and release of the peptide chain transiently oscillate between the ribosome and the soluble cytoplasm [ 1,2 ] . The presence or absence of these molecules in the immediate vicinity of the ribosome will profoundly affect ribosomal function.…”

The protein moieties of animal messenger ribonucleoproteins

“…A more comprehensive recent study has compared the proteins of rabbit reticulocyte polysomal mRNP with initiation factors for protein synthesis [36]. The authors demonstrate that 0.5 m KC1 wash factors contain all the initiation factors but that the mRNA remains bound to the ribosomes together with the proteins of the 14 S mRNP.…”

“…The only suggestion of a functional difference is in the binding of 14 S mRNP to native 40 S ribosomal subunits which have been washed with deoxycholate, a binding which is not found with 9 S mRNA [5]. (The mRNP and mRNA bind equally well to unwashed native 40 S subunits [5, 35,36] . )…”

Globin mRNA Translation on Artemia salina Ribosomes with Components from Friend Leukemia Cells

“…A common characteristic of these heterologous systems is deviation in the CI : fi chain ratio from 1.0, whether or not enzyme fractions from reticulocytes were used. Other reports describe initiation factors that are inactive with [20] or are required for [21,22] translation of globin mRNA. The latter report characterizes a protein from reticulocyte ribosomes which promotes translation of mRNA for a globin chains or tobacco mosaic virus RNA but not p globin chain mRNA or…”