The start site of the Acanthamoeba castellanii ribosomal RNA transcription unit

Abstract

The 39S ribosomal RNA (rRNA) precursor has been isolated from Acanthamoeba castellanii. In vitro capping of the isolated RNA verified that it is the primary transcript and identified the 5' nucleotide as pppA. The position of the 5' coding nucleotide on the rRNA repeat unit sequence was identified using Northern blot, R-loop, and S1 nuclease mapping techniques. Dinucleotide priming of an in vitro transcription system stalled because of low initiating nucleotide concentration revealed that ApA maximally stimulates initiation of transcription. All of these results show that the underlined A in the sequence 5'-TATATATAAAGGGAC (RNA-like strand) coincides with the 5' nucleotide of the primary transcript. This identification is compatible with in vitro transcription experiments mapping the promoter for this transcription unit. The initiation sequences of rRNA genes from 14 species are compared, and a weak consensus for the initiator derived: [Formula; see text].

Figure 1

Analysis of in vitro capped 39S RNA. A. Isolated 39S RNA from Acanthamoeba was capped with α-[³²P]-GTP as described in Materials and Methods, treated as described below, and the products separated by thin layer chromatography. Lane a: untreated. Lane b: bacterial alkaline phosphatase treated. Lane c: P1 nuclease treated. Lane d: P1 nuclease + snake venom phosphodiesterase. Lane e: P1 nuclease + snake venom phosphodiesterase + bacterial alkaline phosphatase treated. B. Capped 39S RNA was hybrid-selected on cloned rRNA and treated with P1 nuclease before separation by PEI thin-layer chromatography.

Figure 2

High resolution S1 nuclease map of 39S RNA. The 161-base Fnu4H I fragment was used for S1 nuclease mapping, and the products were electrophoresed next to a Maxam and Gilbert “C + T” (lane C) and “C > T” (lane D) sequencing ladders of the template DNA strand. Lanes A and B represent the products obtained with increasing S1 nuclease. The T representing the first base in the coding strand is marked with an asterisk, taking into account the 1 1/2 base correction relating the sequencing lanes (which are 3′ phosphorylated) to the S1 nuclease lanes (which are not 3′ phosphorylated).

Figure 3

Mono- or dinucleotide primed synthesis of RNA in a cell free system. In vitro transcription was carried out as described by Paule et al. (1984), except that the concentrations of ATP, GTP, and UTP were set at 10 μM, and 480 μM dinucleoside phosphates were added to some lanes. Lane 1: nucleotides were increased to 600 μM. Lane 2: no additions. Lane 3: ApU. Lane 4: ApG. Lane 5: UpA. Lane 6: ApA. Lane 7: GpA. Lane 8: GpG. Lane 9: GpC. Lane 10: GpU. Lane 11: ATP was increased to 600 μM. Lane 12: GTP was increased to 600 μM. Lane 13: UTP was increased to 600 μM. Lane 14: CTP was increased to 600 μM.

Figure 4

Sequences of the regions surrounding the transcription start sites (RNA-like strands) for Acanthamoeba and 13 other organisms. The first nucleotide of the transcript (+1) is underlined. Transcription initiation sequences are from Acanthamoeba (this paper), Xenopus (Sollner-Webb and Reeder, 1979; Bakken et al., 1982), human (Financsek et al., 1982), mouse (Grummt, 1981), rat (Rothblum et al., 1982; Harrington and Chikaraishi, 1983), Saccharomyces (Klemenz and Geiduschek, 1980), Tetrahymena (Saiga et al., 1982), Neurospora (Niles et al., 1981), Drosophila (Lond et al., 1981), radish (Delcasso-Tremousaygue et al., 1988), maize (McMullen et al., 1986), wheat (Barker et al., 1988), Pisum sativum (Piller et al., 1990), Crithidia (Grondal et al., 1990). The matrix at the bottom compiles the number of times a given nucleotide appears at the position, using 0 to indicate ten times and – to indicate zero times. The consensus shown at the bottom is derived with the following rules: n = any nucleotide; r = purine ⩾75%; upper case letters ⩾67% of the time; lower case letters ⩾50% of the time.