ACTION OF 6‐METHYL PURINE ON RNA SYNTHESIS: INCORPORATION OF THE ADENINE ANALOGUE INTO CODING SEQUENCES AND POLYADENYLATE TRACTS OF MESSENGER RNA IN CHLORELLA

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Abstract
Summary: 6‐methyl purine has been tritiated and, by two‐dimensional paper chromatography, an impurity and two degradation products formed during tritiation have been removed. The authentic 6‐methyl purine has been identified by n.m.r. spectroscopy.Chlorella 211‐8p grown autotrophically in the presence of 1 mM tritiated 6‐methyl purine incorporates radioactivity into RNA. Electrophoretic analysis shows that 45% of the incorporated radioactivity is in RNA fragments with less than 90 bases. There is only trace incorporation into 25s, 18s and 5s rRNA but tRNA accommodates 8% of incorporated label.The bulk of radioactivity found in completed RNA is present in a polydisperse size range of molecules which are identified as mRNA. They are recovered as poly(A)+ RNA by oligo (dT) cellulose binding and are of an appropriate size for a messenger function, since the message which contributes the greatest weight to the total population is of 0·65 × 106mol. wt and could accommodate 677 codons, while electrophoretic analysis of polypeptides from Chlorella shows the size which contributes the greatest weight to be 5 × 104 mol. wt, equivalent to 450 amino acids. This correlates well with the capacity of the predominant message, allowing for the presence of a poly(A) tract.6‐methyl purine is incorporated without modification, since two‐dimensional thin layer chromatography of ribosides derived from mRNA showed that radioactivity co‐chromatographed exclusively with authentic 6‐methyl purine riboside.Poly(A) tracts, prepared from mRNA by digestion of the coding sequences, can be separated from the resulting mononucleotides by electrophoresis. Accurate location of the mononucleotides and the selection of authentic poly(A) segments with oligo (dT) cellulose establishes that tracts of 100 to 200 adenine residues contribute the bulk of poly(A) and so corrects an earlier report that tracts in Chlorella are unusually small. The established sizes of poly(A) tracts and complete messenger molecules indicate a predicted distribution of adenine bases between tract and coding sequences which is close to the observed values of 21 and 89% seen in digests of isolated mRNA. A similarity with poly(A) metabolism in mammalian cells is detected by analysis of the number of tracts of each size, which shows that the smaller sizes are numerically predominant.6‐methyl purine is recovered from both poly(A) tract and coding sequences of mRNA after a 1 h period of autotrophic growth with the inhibitor. At the end of this period one in every three adenines in the tract sequences and one in every 10 adenines in the coding sequences has been substituted by 6‐methyl purine. The incidence in messengers being synthesized in the presence of 6‐methyl purine is probably higher since messengers persisting from before exposure to the inhibitor will give a lower average incidence in the total messenger population.Using minimum estimates of 6‐methyl purine substitution it is calculated that coding sequences for a protein of 40000 mol. wt have only a 1 in 5·3 × 1011probability of containing zero 6‐methyl purine and 99·94% of such messages will contain 12 or more substitutions in the coding sequence. It is concluded that there is negligible formation of normal mRNA by Chlorella 211‐8p in the presence of 1 mM 6‐methyl purine.