Isolation and characterization of polyadenylate-containing RNA from Bacillus brevis

Abstract

A substantial fraction (30--40%) of pulse-labeled RNA from exponentially growing cells of Bacillus brevis contains polyadenylate sequences, as measured by adsorption to oligo(dT)-cellulose. The weight-average length of poly(A) tracts obtained after digestion with pancreatic and T1 ribonucleases is 60 nucleotide residues. Susceptibility to degradation by snake venom phosphodiesterase after ribonuclease degradation indicates that the poly(A) sequences are located near the 3' ends of the RNA chains, but that in 40% of the material at least one internal pyrimidine nucleotide residue intervenes between the poly(A) tract and the 3'-hydroxyl terminus. These pyrimidine nucleotides consist of 65% cytidylate and 35% uridylate residues. In the remaining RNA chains, the poly(A) sequence is directly at the 3'-terminus, but the possibility cannot be excluded that a small fraction of this material may contain a 3'-hydroxyl terminal guanylate residue. The weight-average sedimentation coefficient of poly(A)-containing RNA is 12.5 S, corresponding to a polynucleotide chain length of 800--900 residues. This is in a size range expected for messenger RNA, a possibility which is also supported by the observation that pulse-labeled RNA has a considerably higher poly(A) content than long-term labeled RNA.

Defects in RNA polyadenylation impair both lysogenization by and lytic development of Shiga toxin-converting bacteriophages

“…In fact, it was demonstrated that transcripts derived from over 90 % of ORFs are polyadenylated in E. coli (O'Hara et al, 1995;Mohanty & Kushner, 2006). However, among all RNA molecules appearing due to transcription of a particular gene, only some are polyadenylated, thus, a fraction as small as 2 % of total RNA molecules undergoes this modification under standard laboratory growth conditions (Sarkar et al, 1978). Therefore, enhanced polyadenylation (occurring shortly after prophage induction) can lead to more efficient degradation of transcripts in the WT strain.…”

Quantitative Measurements of Intestinal Ecology by Digital Image Analysed Microscopy

“…The problem here is to find a sequence common to all mRNA. Though it is usually considered a typically eukaryote trait, prokaryote mRNA actually does have a poly-adenylated tail (10,25,26). It is generally shorter than that found in mammalian cells (some tens rather than 200 nucleotides), and though its function has not been clarified completely, it appears to be the signal sequence for ribonucleases which degrade mRNA (34).…”

“…It is generally shorter than that found in mammalian cells (some tens rather than 200 nucleotides), and though its function has not been clarified completely, it appears to be the signal sequence for ribonucleases which degrade mRNA (34). Though to date only four species from the kingdom Bacteria (E. coli, Bacillus subtilis, B. brevis (10,26), and Rhodospirillum rubrum (20)) have been shown to contain poly-A-tails, it is a reasonable assumption that this trait is conserved, and that therefore a poly-T oligonucleotide probe could be used for mRNA quantitation. Figure 7 show poly-T probe derived fluorescence intensity during growth in a batch culture of the Escherichia coli strain JM-101 (Wilkinson and Schut, preliminary data).…”

Polyadenylation of mRNA in bacteria

“…These studies also showed that poly(A) is associated with pulse-labelled RNA, presumably mRNA, whereas long-term-labelled cellular RNA contains a very low level of poly(A) RNA. Susceptibility of the poly(A) tracts to preferential degradation by snake-venom phosphodiesterase indicated that they are primarily located near the 3'-ends of the RNA molecules (Sarkar et al, 1978;Hussain et al, 1982;Gopalakrishna & Sarkar, 1982a;Majumdar & McFadden, 1984). The mean length of the poly(A) tracts, isolated by digesting poly(A) RNA with ribonucleases A and T1 , was estimated from their electrophoretic mobility in polyacrylamide gels and the adenosine : AMP ratio after alkaline hydrolysis, and was found to vary widely amongst bacteria : 13-1 7 nucleotides in Caalobacter crescentus (Ohta et al, 1978), 10-50 nucleotides in E. coli (Nakazato et aZ., 1975), 60 nucleotides in B. brevis (Sarkar e t al., 1978), 49 nucleotides in B. subtilis (Gopalakrishna & Sarkar, 1982a), 10 nucleotides in the archeon M. vamiellii (Brown & Reeve, 1985), and 80 nucleotides in the photosynthetic bacterium Rkodospirillacm rubrum (Majumdar & McFadden, 1984).…”

“…, 1981). Pulse-labelled RNA isolated by this procedure from B. breuis (Sarkar et al, 1978;Gopalakrishna & Sarkar, 1982a), as well as from Bacillas szlbtilis and E. culi B (Gopalakrishna et al, 1981), had a relatively high degree of polyadenylation (1 5-40 %). It seems that earlier reports of very low levels of poly(A) RNA (0.1-0-3%) in the budding bacterium HJVpbumicrubiam sp.…”