The Complete Nucleotide Sequence of a 23‐S rRNA Gene from Tobacco Chloroplasts

Abstract

The nucleotide sequence of a tobacco chloroplast 23-S rRNA gene, including the spacer between it and the 4.5-S rRNA gene, has been determined. The 23-S rRNA coding region is 2804-base-pairs long. A comparison with the 23-S rRNA sequence of Escherichia coli reveals strong homology and further shows a similarity between the chloroplast 4.5-S rRNA and the 3'-terminal region of E. coli 23-S rRNA. However, the 101-base-pair spacer sequence between the 23-S and 4.5-S rRNA genes has little homology with E. coli 23-S rRNA.Elucidation of the structure of ribosomes and the function of large-subunit rRNAs is essentially dependent upon the determination of the primary structure of the rRNA. Up to date the complete nucleotide sequences of the large-subunit rRNA genes (rDNA) from Escherichia coli [l] [20] are well conserved among these large-subunit rRNAs.Tobacco chloroplasts have circular DNAs with molecular weights of around lo8 and their DNAs contain two rDNA clusters, which are located in an inverted order [21-231. We have previously cloned and characterized the rDNAs [24 -271. This rDNA cluster is arranged in the order of genes for 16-S, 23-S, 4.5-S and 5-S rRNAs and these four rDNAs are coded for by the same strand and the transcription proceeds from the 16-S to the 5-S rRNA gene [25,26]. The sequences of the DNAs coding for 4.5-S and 5-S rRNAs and their surrounding regions [28] and that of the 16-S rRNA [29] and its 5'-flanking region [30] have been determined previously. In this paper, we present the nucleotide sequence of a 23-S rRNA gene including the spacer between it and that for 4.5-S rRNA from tobacco chloroplasts and compare it with other large-subunit rDNAs. MATERIALS AND METHODS MaterialsTobacco (Nicotiana tabacum var. Bright Yellow 4) chloroplast 23-S rRNA was prepared as described [24] Preparation of DNA Fragments and Sequencing Recombinant plasmid pTCP243 1261 was propagated in Escherichia coliHBIO1 cells and the plasmid DNA was isolated by the cleared lysate method [33]. The DNA was digested with HindIII, XhoI or EcoRI + HindIII, and the digests were separated on 5 % polyacrylamide gels (14 x 11 x 0.5 cm). The DNA fragments containing the 23-S rRNA gene were recovered from the excised gel pieces by electrophoresis. The fragments were purified by DEAE-Sephacel chromatography [29]. DNA fragments were labeled at the 5' ends with [Y-~'P]ATP and T4 polynucleotide kinase after alkaline phosphatase treatment as described by Maxam and Gilbert [34]. The 3'-terminal labeling was carried out by using [IX-'~P]~CTP and T4 DNA polymerase as described by Challberg and Englund [35]. Labeled fragments were separated on 6 % or 8 polyacrylamide gels (37 x 28 x 0.05 cm). Strand separation was performed on 5 % polyacrylamide gels (52 x 28 x 0.05 cm). DNA fragments were eluted from the polyacrylamide gel by diffusion as described by Maxam and Gilbert [34]. Nucleoside-specific chemical cleavages (dG, dA>dC, dT+dC and dC) were performed according to the method of Maxam and Gilbert [34].Partial cleavage products were analyzed on...

Structure and function of ribosomal RNA

“…The most extreme example is the ribosome of Crithidia fasciculata, whose large subunit contains no less than four extra small RNA species in addition to 5S and 5.8S RNA (Schnare et al, 1983), although it has yet to be established whether all of these have 23S RNA counterparts. In contrast to this type of extra processing event, it has recently been proposed that some fungal mitochondrial ribosomes (which, as noted above, lack 5S RNA) may have actually incorporated a 5S-like sequence into their large subunit RNA molecules Dron et al (1982) Brosius et al (1978, Carbon et al (1978 Carbon et al (1981) Kop et al (1984a) Iwami et al (1984) 12S Eperon et al (1983) 16S Van Etten et al (1980) 16S Saccone et al (1981) 16S Eperon et al (1980) 16S Anderson et al (1982) 20Sb Seilhammer & Cummings (1981) 20Sb Seilhammer et al (1984b) 21S Sor & Fukuhara (1983) 23S Kochel & Kuntzel (1982) 26S Dale et al (1984) 23SC Edwards & Kossel (1981) 23SC Takaiwa & Sugiura (1982) 23S Brosius et al (1980), Branlant et al (1981) 23S Kumano et al (1983, Douglas & Doolittle (1984) 23S Kop et al (1984b) 16S Gupta et al (1983) 16S Leffers & Garrett (1984) McCarroll et al (1983) Takaiwa et al (1984) Messing et al (1984) Rubtsov et al (1980 Hadjiolov et al (1984), Chan et al (1983) There are a small number of modified nucleotides in ribosomal RNA. In E. coli, the 16S rRNA contains nine methylated bases (Carbon et al, 1979) and the 23S rRNA ten methylated bases and three pseudouridine residues .…”

Structure and evolution of the 4.5–5S ribosomal RNA intergenic region fromGlycine max(Soya bean)

“…Sequence studies indicate that all of the chloroplast rRNAs are transcribed as a common larger precursor molecule, the sequences being arranged in the order 5'-16S-23S-4.5S-5S-3' (5-7). As ribosomal RNA from many origins have been sequenced, comparisons have clearly shown (5)(6)(7)(8) that the 4.5S rRNA sequence is not unique to plant chloroplasts; it is actually equivalent to the 3' end of the eubacterial 23S rRNA and is the result of an extra cleavage or processing step in the chlorplast rRNA precursor.…”

“…The 3' end of the chloroplast rRNA precursor is a particularly good model systems for studies on rRNA maturation because five junctions (3' end of the 23S rRNA, 5' and 3' ends of the 4.5S rRNA, 5' and 3' ends of the 5S rRNA) are present within a relatively short sequence. Despite this, the complete nucleotide sequence for all the spacers regions is known in only two plants, tobacco and duckweed (7,(10)(11)(12) although a number of sequences for the mature 4.5, 5S and 23S rRNA sequences or at least one of the ITS regions have been determined (e.g. 4,[13][14][15].…”

The Plastid Ribosomal Proteins

“…The 4.5 S rRNA essentially represents the corresponding 3Ј end portion of bacterial 23 S rRNA (73). The sum of tobacco 23 S rRNA (2804 nucleotides) and 4.5 S rRNA (103 nucleotides) is just three nucleotides larger than E. coli 23 S rRNA (2904 nucleotides).…”