Isolation of a protein fraction from reticulocyte ribosomes required for de novo synthesis of hemoglobin

Abstract

A fraction (I fraction) was prepared by extracting IX-reticulocyte ribosomes (unwashed) with 0.5 m KCl. This fraction is required for de novo synthesis of hemoglobin by various ribosomal preparations; however, it is not required for the completion of nascent chains. The active component(s) in the I fraction is protein in nature, since it is inactivated by pronase, trypsin, and N-ethylmaleimide (NEM) but is not affected by T 1 RNase. The active component(s) of the I fraction appears to have a molecular weight of at least 100,000. It appears to be involved in some step of protein synthesis following the formation of aminoacyl-tRNA but prior to the steps requiring the transfer enzymes (TF-1 and TF-2). The 1X-ribosomes are broken down into particles which sediment slower than 80S monosomes by the salt wash. In spite of this drastic treatment of 1X-ribosomes, the hemoglobin mRNA remains associated with the ribosomes.