The topographical order of 18 S and 20 S ribosomal ribonucleic acids within the 45 S precursor molecule

Abstract

Nucleoli isolated from the Novikoff rat tumor were used to study the biosynthesis and methylation of ribosomal RNA in vitro . Nucleolar nascent preribosomal RNA was labeled in limited regions with methyl - 3 H groups by incubation of nucleoli with S -adenosyl- l -[ methyl - 3 H]methionine under conditions which minimize degradation and further elongation of the RNA chains. The labeled nucleolar RNA was subsequently extracted by phenol treatment and fractionated by sucrose gradient centrifugation into 0 to 20 S, 20 to 30 S, and 30 to 50 S fractions. This produces an analogous effect of dividing the 45 S preribosomal RNA into three regions as far as the ribose methylation is concerned; the first region represents 17% of the 45 S RNA from the 5′-end (0 to 20 S), the second region represents 31% of the 45 S RNA from the middle segment (20 to 30 S), and the third region represents 52% of the 45 S RNA from the 3′-end (30 to 50 S). The methylated nucleotide patterns (both base and ribose) of these fractions were analyzed and compared with the patterns of the ribosomal RNAs. The ribose methylation patterns of the 0 to 20 S fraction resembled more closely those of 18 S ribosomal RNA, and the ribose methylation patterns of the 30 to 50 S fraction resembled more closely those of 28 S ribosomal RNA. A marker alkali-stable trinucleotide, probably UmpGmpU, known to be present only in 28 S ribosomal RNA, was found to be labeled and concentrated in the 30 to 50 S fraction under these conditions. Preferential inhibition by 0·5 m -KCl of the methylation of nascent RNA sedimenting as the lower molecular weight fraction led to the production of methylated patterns resembling more closely those of 28 S ribosomal RNA. It is concluded from these data that 18 S ribosomal RNA is located nearer to the 5′-end and 28 S ribosomal RNA is nearer to the 3′-end of the 45 S preribosomal RNA. The results also demonstrate the validity and utility of these procedures for the study of ribosomal RNA synthesis in vitro .