Amino acid incorporation on plant ribosomes: The transfer system

Pre-formed Vicia faba phenylalanyl-tRNA was active in a TYMV-RNA-directed Transfer System, whereas a similar tRNA preparation from yeast was not. Thus, lack of charging of yeast tRNA by enzymes from Phaseolus was not the only reason why yeast tRNA would not function in this Transfer System. In the poly U-directed Transfer System; where both types of tRNA were active, the pH and ionic parameters governing the reaction with yeast tRNA were more stringent.     

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.     

Amino Acid incorporation by wheat chloroplasts

Isolated chloroplasts from wheat leaves incorporate radioactive amino acids into protein. Both physiological and biochemical evidence show that contaminating bacteria are not responsible for the activity. Activity is best in plastids from 5-day-old or younger seedlings; a sharp drop usually occurs by day 6 or 7. The system requires added adenosine triphosphate, guanosine triphosphate and Mg⁺⁺, and is inhibited by ribonuclease, puromycin and chloramphenicol. Preliminary evidence is presented that polyribosomes are present in the young leaf chloroplast fraction. Half of the protein that is formed in a 20-minute incubation is released in soluble form.     

Synthesis and characterization of a spin-labeled aminoacyl transfer ribonucleic acid

Unfractionated yeast transfer ribonucleic acid (tRNA) was reacted in aqueous acetone solution with the sulfhydryl spin-labeling reagnent, N-(l-oxyl-2,2,5,5-tetramethyl-3-Pyrrolidinyl)iodoacetamide. Whereas tRNA stripped of amino acids reacted only slowly, there were sites on tRNA charged with cysteine which combined rapidly with the reagent. The latter class of spin-label was quickly cleaved from the tRNA upon incubation in mildly alkaline solution (pH 8.0), suggesting that it was attached to the cysteinyl side chains. The paramagnetic resonance spectrum of column-purified spin-labeled cysteinyl tRNA showed that the spin-label was partially immobilized as a result of its interaction with the tRNA. When the tRNA was slowly heated, an abrupt increase occurred in the rotational mobility of the paramagnetic amino-acid side chain.