Amino acid incorporation by ribosomes and polyribosomes from wheat chloroplasts

Sucrose-gradient and analytical ultracentrifugation showed that chloroplast polyribosomes from 4-day-old seedlings had mono-, di-, tri-, tetra- and traces of penta-ribosomes, in contrast with those from 7-day-old seedlings in which only the mono-, di- and traces of tri-ribosomes were present. Without Mg(2+) the polyribosomes dissociated into ribosomal subunits. The rate of l-[U-(14)C]phenylalanine incorporation was threefold greater for preparations from 4- than from 7-day-old seedlings. Incorporation by the latter was stimulated by polyuridylic acid. The rates of incorporation were similar whether the reaction mixture contained chloroplast or wheat-germ transfer RNA and amino acid synthetases purified on methylated albumin-on-kieselguhr and Sephadex G-75 columns respectively. The cofactor requirement was the same as for isolated intact chloroplasts. Osmotic rupture of chloroplasts with and without Triton X-100 revealed the presence of free and bound ribosomes. Free single ribosomes isolated by osmotic shrinkage or prepared by pancreatic ribonuclease digestion of chloroplast polyribosomes had negligible incorporation activity. This activity was increased by washing or by polyuridylic acid, but was still only a fraction of that given by polyribosomes. A comparison of incorporation activity of chloroplast polyribosomes with those from the surrounding cytoplasm showed the former to be 20 times more active.     

Membrane-bound polyribosomes in HeLa cells: association of polyadenylic acid with membranes

Polyadenylic acid of membrane-bound polyribosomes is shown to be associated with rapidly sedimenting membrane structures. Most of this poly(A) remains attached to membranes after extensive degradation of polyribosomal messenger RNA with pancreatic ribonuclease. Previously, it was shown that exposure to EDTA removes up to 40% of the membrane-associated mRNA. In our experiments, 57% of the membrane-associated poly(A) still sediments with membrane structures after treatment with pancreatic ribonuclease followed by the addition of EDTA. This indicates that the association of about 60% of the membrane-bound poly(A) is EDTA-resistant, while the remainder is labile after removal of magnesium ions. Reconstruction experiments suggest that poly(A) from detergent-treated, membrane-derived polyribosomes is not trapped by other membrane structures. The poly(A)-containing RNA fragment that remains associated with the membranes after pancreatic ribonuclease treatment is shown to be a single peak at about 7 S, or about the size of cellular poly(A). Thus, the attachment site is almost pure poly(A). The poly(A)-containing, membrane-bound mRNA appears to be of a larger average size than total cellular poly(A)-containing RNA, as judged by its greater sedimentation value.     

The discovery of polyribosomes

By the early 1960s, evidence had accumulated that proteins were synthesized from special RNA copies of genes, named "messenger RNAs" (mRNAs), not directly from the stable RNAs found in the ribosomes of the cytoplasm. Yet, precisely how the protein chains were assembled along the RNA and, in particular, the relationship between the mRNAs and the ribosomes during protein synthesis, was obscure. In this account, I discuss how my laboratory found that multiple ribosomes traverse each mRNA, yielding the structures known as polysomes. This work led on to the first physical determination of the coding ratio, new insights into how protein chains are initiated, and an early suggestion that chloroplasts and mitochondria in eukaryotic cells might ultimately have been derived from symbiotic bacteria.     

On the fine structure of polyribosomes

Cell and Tissue Research - The ultrastructural morphology of ribosomes was studied in tissue sections of rat uterus using different fixatives (acrolein, formaldehyde, acetic acid, methanol-acetic...