The complete nucleotide sequence of the 25 S rRNA gene from one rDNA repeating unit of Saccharomyces cerevisiae has been determined. The corresponding 25 S rRNA molecule contains 3392 nucleotides and has an estimated relative molecular mass (Mr, Na-salt) or 1.17 x 10(6). Striking sequence homology is observed with known 5'- and 3'-end terminal segments of L-rRNA from other eukaryotes. Possible models of interaction with 5.8 S rRNA are discussed.

A mutant of E. coli, isolated by Kindler and Hofschneider as a strain defective in RNase III activity, forms a 30S precursor of ribosomal RNA ("30S pre-rRNA"). The half-life of the 30S pre-rRNA in growing cells at 300, estimated by the rate of specific 3[H]uridine incorporation, is about 1 min. In rifampicin-treated cells, the RNA is metabolized to mature rRNA with a halflife of about 2 min.The 30S pre-rRNA has been highly purified. DNA-RNA hybridization tests demonstrate that it contains both 16S and 23S rRNA sequences. Also, in cultures treated with rifampicin, the cleavage products of radioactive 30S prerRNA include 25S and 17.5S RNA species, destined to become 23S and 16S rRNA. Thus, each 30S chain probably contains one 16S and one 23S RNA sequence, as well as additional sequences. Two independent techniques indicate that the additional portions account for about 27% of the total length: (1) By comparison to the sedimentation rate and electrophoretic mobility of marker RNAs, the 30S pre-RNA has an apparent molecular weight of 2.3 X 106 ± 5%G, or 28%o more than the sum of 16S and 23S rRNA;(2) 27%o of the 30S pre-rRNA is not competed away from hybridization by mature 16S and 23S rRNA.Thus, bacteria appear to make a pre-rRNA similar in some respects to that observed in eukaryotes; though in normal E. coli cells, the pre-rRNA is ordinarily cleaved endonucleolytically during its formation. AB105, isolated by Hofschneider and his collaborators (1), was originally characterized as a strain of Escherichia coli that grew poorly even in rich medium, and yielded extracts which showed little or no attack on the double-stranded replicative form of RNA from R17 phage. Such endonucleolytic attack is attributed to RNase III (2).One suggested process in which endonuclease function might be required is the formation of 23S, 16S, and perhaps 5S RNA from longer transcripts, possibly even starting from a single initiation point (3, Pace, N. R., review in preparation). In growing bacteria, this process has been inferred only from indirect experiments that sometimes allowed conflicting interpretations (4, Pace, N. R., review in preparation). In AB105, the process is slowed and a precursor containing both 16S and 23S rRNA becomes directly observable. 30S precursor to ribosomal RNA, pre-rRNA, was purified by two successive zonal sedimentation runs. 38-ml 10-30% sucrose gradients were used in the SW27 rotor of the L2-65B Spinco ultracentrifuge (see Fig. 4). The buffer in the gradient was 0.1 Al Tris-acetate (pH 7.5) containing 0.5% sodium dodecyl sulfate, which permitted centrifugation at 4°without salt precipitation. Centrifugation in each run was for 23 hr at 25,000 rpm. 0.75-ml Fractions were collected by pumping from the bottom of the tubes. The fractions from the second gradient containing the 30S pre-rRNA (as in Results, Fig. 4) were again precipitated with ethanol.For DNA-RNA hybridization analyses, the purified 30S Unlabeled 16S and 23S rRNA for use as competitors in hybridization trials (Fig. 6) were prepared by p...

The 5'-terminal coding sequence for the 37 S precursor to rRNA of Saccharomyces cerevisiae is identified by reverse transcriptase extension and protection mapping with nuclease S1. The sequence of a 419 bp rDNA fragment containing the transcription initiation site and its adjacent region is determined.