Identification and comparative analysis of the chloroplast alpha-subunit gene of DNA-dependent RNA polymerase from seven Euglena species

Abstract

When the sequence of the Euglena gracilis chloroplast genome was reported in 1993 the alpha-subunit gene (rpoA) of RNA polymerase appeared to be missing, based on a comparison of all putative reading frames to the then known rpoA loci. Since there has been a large increase in known rpoA sequences, the question of a Euglena chloroplast rpoA gene was re-examined. A previously described unknown reading frame of 161 codons was found to be part of an rpoA gene split by a single group III intron. This rpoA gene, which is highly variable from species to species, was then isolated and characterized in five other euglenoid species, Euglena anabaena, Euglena granulata, Euglena myxocylindracea, Euglena stellata and Euglena viridis, and in the Astasia longa plastid genome. All seven Euglena rpoA genes have either one or three group III introns. The rpoA gene products in Euglena spp. appear to be the most variable in this gene family when compared to the rpoA gene in other species of bacteria, algae and plants. Additionally, Euglena rpoA proteins lack a C-terminal domain required for interaction with some regulatory proteins, a feature shared only with some chlorophyte green algae. The E.gracilis rpoA gene is the distal cistron of a multigene cluster that includes genes for carbohydrate biosynthesis, photosynthetic electron transport, an antenna complex and ribosomal proteins. This study provides new insights into the transcription system of euglenoid plastids, the organization of the plastid genome, group III intron evolution and euglenoid phylogeny.

Figure 1

Schematic diagram of the ycf9–rpoA–trnS region. Primers P1 and P2 were used for amplification of this region. Black boxes represent the genes for ycf9, rpoA and trnS. Group III introns are indicated by red lollipops. (A) The previously reported E.gracilis gene organization (4) and the primers used for PCR and RT–PCR. (B) The E.gracilis rpoA locus as characterized in this study. (C) The postulated A.longa rpoA coding locus. (D) The rpoA coding loci for five Euglena species. One intron is present in each of E.gracilis and A.longa. Two additional introns are present in E.anabaena, E.granulata, E.myxocylindracea, E.stellata and E.viridis.

Figure 2

An rpoA amino acid sequence alignment using PILEUP (GCG Sequence Analysis Package v.8.0). Conserved amino acids in Euglena spp. are indicated by a black background, while similar amino acids are indicated by a yellow background. Similar amino acids are defined as E and D, F and Y, R and K, L, V, I and M, and S and T. Locations of introns are indicated by vertical arrows. The amino acids that were chosen randomly for the Euglena consensus sequence are shown outlined. The consensus of the N-terminal sequence from 34 species of bacteria, algae and land plants is shown. Conserved amino acids between the two consensus sequences are shown and similar amino acids are indicated by a + sign. The secondary structure of the αNTD is indicated schematically above the α sequences; helices H1–H3 are indicated by rectangles; strands S1–S11 are indicated by arrows. Black dots denote residues that participate in the hydrophobic core of the α dimer interface and green dots indicate mutations that cause defects in binding to other subunits that were previously reported for E.coli (14).

Figure 2

An rpoA amino acid sequence alignment using PILEUP (GCG Sequence Analysis Package v.8.0). Conserved amino acids in Euglena spp. are indicated by a black background, while similar amino acids are indicated by a yellow background. Similar amino acids are defined as E and D, F and Y, R and K, L, V, I and M, and S and T. Locations of introns are indicated by vertical arrows. The amino acids that were chosen randomly for the Euglena consensus sequence are shown outlined. The consensus of the N-terminal sequence from 34 species of bacteria, algae and land plants is shown. Conserved amino acids between the two consensus sequences are shown and similar amino acids are indicated by a + sign. The secondary structure of the αNTD is indicated schematically above the α sequences; helices H1–H3 are indicated by rectangles; strands S1–S11 are indicated by arrows. Black dots denote residues that participate in the hydrophobic core of the α dimer interface and green dots indicate mutations that cause defects in binding to other subunits that were previously reported for E.coli (14).

Figure 3

The E.gracilis chloroplast rbcL–rpoA operon. The Euglena chloroplast gene is encoded in the rbcL operon, which is ∼10.6 kb in size. The arrow indicates the direction of transcription. Boxes denote genes of the operon. Large blue lollipops indicate Group II introns, while small red lollipops indicate Group III introns.

Figure 4

Phylogenetic NJ tree inferred from a rpoA sequence comparison. Branch lengths are proportional to the expected mean number of substitutions per site along the branch, as quantified by the scale bar. The tree was generated using CLUSTAL X (GCG Sequence Analysis Package v.8.0). Boostrap numbers for the 1000 boostrap replications conducted are indicated.