Polyadenylation is an important structural feature of mRNAs in both eucaryotes and eubacteria. The poly(A) tracts, as well as the RNA molecules of which they are a part, are however quite different in the two groups. Eubacterial polyadenylated [poly(A)+] RNAs exhibit a wide range of sizes and usually have only short poly(A) tracts (3-6, 9). They are unstable and therefore constitute only a small percentage of the total cellular RNA (3-5, 9, 11). We recently described the isolation and characterization of poly(A)+ RNA molecules from the methanogenic archaebacterium Methanococcus vannielii (1) and found that they share the eubacterial properties of short 3' poly(A) tracts, short half-lives and a broad size distribution. During our study of the methanogenic archaebacterium, somewhat conflicting results concerning polyadenylation of RNA in the halophilic archaebacterium Halobacterium halobium were published. The mRNA encoding bacterio-opsin in H. halobium does not appear to be poly(A)+ (2), and yet a preliminary report was published describing very long, eucaryotic-like poly(A) tracts in RNA molecules isolated from H. halobium and from the thermoacidophilic archaebacterial genera Thermoplasma and Sulfolobus (10). We have now extended our studies to investigate H. halobium and report here that poly(A) sequences are present in H. halobium RNAs, but that the poly(A) tracts, although present in relatively large amounts, are much shorter than those normally found in eucaryotic mRNAs.Poly ( 10,000-fold excess of unlabeled uridine was added, and the amount of poly(A)+ RNA was determined at 1, 2.5, 5, 10, 20, 30, 45, and 60 min after the labeling period. The results obtained showed that the average half-life of poly(A)+ RNA molecules in H. halobium at 37°C was 4.6 min, which is equivalent to 1/60 of a generation time.The size distribution of radioactively labeled poly(A)+ RNA from H. halobium was determined by electrophoresis of denatured RNA through agarose gels, followed by fractionation of the gel and scintillation counting of each gel slice. Poly(A)+ RNA molecules were found to range from 0.5 to 3.0 kilobases, averaging 1.1 kilobase in length (Fig. 1).This size range is consistent with the presence of both monocistronic and polycistronic mRNAs and is similar to the range of sizes of mRNAs found in M. vannielii and in several eubacterial species (1, 5, 9, 11).The amount of poly(A), as a fraction of the total cellular and separated by electrophoresis through a 1.8% agarose gel. The locations of stable RNAs were determined by examination of adjacent lanes of the gel containing total RNA after staining with acridine orange. The gel in the appropriate lane was sliced into fractions, and the radioactivity in each fraction was determined.

RNA molecules have been isolated from Methanococcus vannielii by oligodeoxythymidylate-cellulose affinity chromatography at 4C. Approximately 16% of the label in RNA isolated from cultures allowed to incorporate [3H]uridine for 3 min at 37C was poly(A)+ RNA. In contrast, less than 1% of the radioactivity in RNA labeled over a period of several generations was contained in poly(A)+ RNA molecules. Electrophoretic separation of poly(A)+ RNA molecules showed a heterogeneous population with mobilities indicative of sizes ranging from 900 to 3,000 bases in length. The population of poly(A)+ RNA molecules was found to have a half-life in vivo of approximately 12 min. Polyadenylate [poly(A)] tracts were isolated by digestion with RNase A and RNase T, after 3' end labeling of the poly(A)+ RNA with RNA ligase.

No abstract