Bernadette Pace

The chromatography of RNA and oligoribonucleotides on boronate-substituted agarose and polyacrylamide

Abstract Several boronate-containing supports for the separation of oligo- or polyribonucleotides, on the basis of their content vs lack of cis -diol groups, have been described in the literature; boronate cellulose (DBAE-cellulose) and boronate polyacrylamide are available commercially. Boronate cellulose binds macromolecular RNA, but often is not useful for trace amounts of material because of nonspecific adsorption. We have found that boronate polyacrylamide and boronate agarose (not yet commercially available) both display very low levels of nonspecific adsorption. Boronate polyacrylamide specifically binds cis -diol-containing mononucleotides or short oligonucleotides, but does not retain polyribonucleotides sufficiently tenaciously for preparative use. In contrast, boronate agarose proved suitable for the fractionation of trace amounts of macromolecular RNA, but not for mononucleotides.

The nucleotide sequence of chicken 5S ribosomal RNA.

SummaryThe nucleotide sequence of the 5S ribosomal RNA of somatic cells of the chicken (Gallus gallus) differs from that of other vertebrates thus far examined. Besides base substitutions, alterations include two nucleotide deletions and two additions.

Ribosomal RNA terminal maturase: Ribonuclease M5 from Bacillus subtilis

Publisher Summary The ribosomal RNAs (rRNA) of all cells are acted upon by several RNA processing nucleases and nucleotide-modifying enzymes during the formation of the mature ribosome. The general requirement of the terminal maturases for RNP substrates is best evidenced by the fact that the immediate precursors of the mature rRNAs accumulate in cells treated with inhibitors of protein synthesis. The proteins required for the formation of productive substrates for the terminal rRNA maturases probably are generally ribosomal proteins, known to associate with the nascent rRNAs during transcription. The terminal rRNA maturases are rare: each B. subtilis cell probably contains no more than about I00 molecules of RNase M5. The extensive purification of rare enzymes such as RNase M5 inevitably results in low solution concentrations of protein, a condition that is considered to predispose enzymes to denaturation and loss of activity. Most of the reported studies of RNase M5 used enzyme at this level of purity. Preparations are greatly enriched in RNase M5 activity and free of other nucleases.

The Interaction of RNase M5 with a 5S rRNA Precursor

As is evident from several other papers in this volume, considerable information regarding the posttranscriptional processing of tRNA is accumulating. Although most RNA molecules, in both prokaryotes and eukaryotes, are the products of more or less extensive posttranscriptional metabolism, detailed studies of these processes have been possible only with tRNA and 5S rRNA of Bacillus subtilis. Although not directly involved with tRNA, studies of the processing of 5S rRNA are relevant in this area of study in that much of this methodology is immediately applicable to the study of the maturation of the precursors of tRNA. Moreover, there is the common goal of understanding the nature of protein-polynucleotide interactions. In brief, we have isolated the endonuclease responsible for the terminal maturation of 5S rRNA in B. subtilis and devised procedures for altering the precursor RNA substrate to explore the polynucleotide recognition elements utilized by this highly specific enzyme. THE MATURATION OF B. SUBTILIS 5S rRNA Because of its simple structure (about 120 nucleotides long), 5S rRNA is useful for exploring the details of posttranscriptional RNA metabolism. At the outset of these studies, we felt that the 5S rRNA of Escherichia coli was not likely to be a suitable model, since the immediate precursor of 5S rRNA in this organism is maximally only 3 nucleotides larger than the mature form (Monier et al. 1970) and such limited precursor-specific length might not be of functional significance. We therefore undertook a search for an organism utilizing more complex 5S rRNA metabolism than that...