Molecular Biology and Evolution

MBE Advance Access originally published online on March 13, 2006
Molecular Biology and Evolution 2006 23(6):1169-1179; doi:10.1093/molbev/msk001

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Research Article

Homologs of Mitochondrial Transcription Factor B, Sparsely Distributed Within the Eukaryotic Radiation, Are Likely Derived from the Dimethyladenosine Methyltransferase of the Mitochondrial Endosymbiont

Timothy E. Shutt and Michael W. Gray

Program in Evolutionary Biology, Canadian Institute for Advanced Research, Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, Canada

E-mail: m.w.gray{at}dal.ca.

	Abstract

TOP Abstract Introduction Materials and Methods Results Discussion Conclusions Supplementary Material Acknowledgements References

 
Mitochondrial transcription factor B (mtTFB), an essential component in regulating the expression of mitochondrial DNA-encoded genes in both yeast and humans, is a dimethyladenosine methyltransferase (DMT) that has acquired a secondary role in mitochondrial transcription. So far, mtTFB has only been well studied in Opisthokonta (metazoan animals and fungi). Here we investigate the phylogenetic distribution of mtTFB homologs throughout the domain Eucarya, documenting the first examples of this protein outside of the opisthokonts. Surprisingly, we identified putative mtTFB homologs only in amoebozoan protists and trypanosomatids. Phylogenetic analysis together with conservation of intron positions in amoebozoan and human genes supports the grouping of the putative mtTFB homologs as a distinct clade. Phylogenetic analysis further demonstrates that the mtTFB is most likely derived from the DMT of the mitochondrial endosymbiont.

Key Words: mitochondrial transcription • mtTFB • endosymbiont • dimethyladenosine methyltransferase

	Introduction

TOP Abstract Introduction Materials and Methods Results Discussion Conclusions Supplementary Material Acknowledgements References

 
Mitochondria contain their own genome, which in different eukaryotic lineages is greatly reduced and extremely variable in size and gene content compared to its -proteobacterial ancestor (Gray, Burger, and Lang 1999; Burger, Gray, and Lang 2003). In most eukaryotes, mitochondrial genes are transcribed by a single-subunit RNA polymerase (ssRNAP), which appears to have originated from a T-odd–like bacteriophage (Masters, Stohl, and Clayton 1987; Cermakian et al. 1996; Hedke, Borner, and Weihe 1997; Grams et al. 2002). The ssRNAP of T7 phage is a functional monomer and does not require any accessory proteins (Cheetham and Steitz 2000). However, this does not appear to be the case for the mitochondrial ssRNAP of yeast and metazoan animals.

In yeast (Saccharomyces cerevisiae), the nucleus-encoded mitochondrial transcription factor 1 (SC-mtTFB, specified by the MTF1 gene; Lisowsky and Michaelis 1988) was initially thought to resemble bacterial sigma factors (Jang and Jaehning 1991). (Here we use the transcription factor nomenclature proposed by Xu and Clayton [1992].) Later, three-dimensional structures showed that sc-mtTFB is instead homologous to the Escherichia coli RNA adenine dimethyltransferase KsgA (Schubot et al. 2001; O'Farrell, Scarsdale, and Rife 2004).

Homologs of KsgA are found in all three domains of life and are responsible for the highly conserved dimethylation of two adjacent adenine residues (A1518 and A1519 in E. coli) in the small subunit ribosomal RNA (SSU rRNA). This methylation is not essential in bacteria; in fact, lack of methylation confers resistance to the antibiotic kasugamycin (Hesler, Davies, and Dahlberg 1971). Intriguingly, the yeast mitochondrial SSU rRNA is one of the few exceptions known in which dimethylation of the two adjacent A residues does not occur (Klootwijk, Klein, and Grivell 1975), although information regarding SSU rRNA modifications in other mitochondrial systems is sparse.

Bioinformatic analyses have identified human transcription factors B1 and B2 (h-mtTFB1 and h-mtTFB2, encoded by the TFB1M and TFB2M genes, respectively) as homologs of sc-mtTFB (Falkenberg et al. 2002; McCulloch, Seidel-Rogol, and Shadel 2002). The h-mtTFB1 protein has been shown to have dual function in that it is also able to methylate E. coli SSU rRNA at the same sites as KsgA (Seidel-Rogol, McCulloch, and Shadel 2003).

So far, homologs of mtTFB have only been studied in the opisthokonts (Opisthokonta), a eukaryotic supergroup comprising metazoan animals and fungi (Simpson and Roger 2004; Keeling et al. 2005); thus, it is unclear whether mtTFB is specific to this lineage or whether it is a eukaryote-wide feature of the mitochondrial transcription apparatus. In this study, we identify the first homologs of mtTFB outside of the opisthokonts. Notably, homologs of mtTFB are absent (or not easily identifiable) in several completely sequenced eukaryotic genomes. In addition, phylogenetic analysis of identified mtTFB sequences shows that the mtTFB genes are not derived from the eukaryotic dimethyladenosine methyltransferase (DMT) but rather are specifically related to the DMT of -proteobacteria and are thus likely to have been derived in evolution from the genome of the eubacterial endosymbiont that gave rise to mitochondria.

	Materials and Methods

TOP Abstract Introduction Materials and Methods Results Discussion Conclusions Supplementary Material Acknowledgements References

 
Nucleic Acids
Samples of genomic DNA from Acanthamoeba castellanii (ATCC 30010) and Hartmannella vermiformis (ATCC 50236) were kindly provided by A. J. Lohan. Samples of total RNA from A. castellanii and H. vermiformis were from A. J. Lohan and C. E. Bullerwell, respectively.

Initial Characterization of mtTFB Gene Sequences from A. castellanii and H. vermiformis
A portion of the cDNA sequence encoding A. castellanii mtTFB was identified from expressed sequence tag (EST) data generated by the Protist EST Program and available through PEPdbPub (http://amoebidia.bcm.umontreal.ca/public/pepdb/agrm.php). This sequence was used as a query for Blast analysis to identify the putative mtTFB sequence in the genome of Dictyostelium discoideum. An initial alignment of putative A. castellanii and D. discoideum mtTFB sequences with metazoan mtTFB1 and mtTFB2 sequences was used for design of degenerate primers targeted to highly conserved regions.

Degenerate Polymerase Chain Reaction
An internal fragment of the H. vermiformis mtTFB genomic sequence was amplified by two successive rounds of polymerase chain reaction (PCR) with degenerate primers TFB-CH-2 and TFB-CH-4 (100 pmol each; see table 1 for primer sequences) using Invitrogen (Carlsbad, Calif.) Taq DNA polymerase and recommended reagent concentrations. The first round of PCR used 100 ng of genomic DNA, while the second round used 1 µl of the 50-µl PCR reaction from the first round. PCR cycling reactions were performed using a PerkinElmer GeneAmp PCR system 2400 under the following conditions: 94°C for 3 min; 35 cycles at 94°C for 30 s, 55°C for 30 s, 50% temperature ramp to 72°C for 60 s; and 72°C for 10 min. A PCR product of approximately 550 bp in length was isolated from an agarose gel using the Sephaglas BandPrep Kit (Amersham Pharmacia Biotech. Piscataway, N.J.). The recovered DNA was cloned into the pCR 2.1-TOPO vector using the TOPO TA Cloning Kit (Invitrogen). DNA sequencing was performed with an automated ABI Prism 377 DNA sequencer.

View this table: [in this window] [in a new window]   Table 1 Oligonucleotide Primers

 
Restriction-Site Polymerase Chain Reaction
The 5' and 3' ends of the H. vermiformis mtTFB gene and the 5' end of its A. castellanii counterpart were obtained using a modified restriction-site polymerase chain reaction (RS-PCR) approach (Weber, Bolander, and Sarkar 1988; Sarkar, Turner, and Bolander 1993) employing the primers listed in table 1. The first round of RS-PCR used 100 ng of genomic DNA as template with 2 pmol of sequence-specific primer and a combination of RS-PCR primers (20 pmol each), under the following cycling conditions: 3 min at 94°C; 35 cycles of 30 s at 94°C, 120 s at 50°C, 120 s at 72°C; and 10 min at 72°C. A second round of RS-PCR was then performed using 1 µl of the 50-µl PCR from the first round with 20 pmol of a nested-specific primer and the RS-PCR for primer under the following conditions: 3 min at 94°C; 35 cycles of 30 s at 94°C, 120 s at 55°C, 120 s at 72°C; and 10 min at 72°C. DNA was isolated from gels, cloned, and sequenced as above.

Genomic PCR of A. castellanii mtTFB
In order to obtain information regarding intron content, the complete sequence of the A. castellanii mtTFB gene was obtained by PCR from genomic DNA using primers AcTFB-5' end-for and AcTFB-3' end-rev under the following conditions: 3 min at 94°C; 35 cycles of 30 s at 94°C, 60 s at 55°C, 60 s at 72°C; and 10 min at 72°C. A PCR product of approximately 1,600 bp in length was isolated from an agarose gel, cloned, and sequenced as above.

5' Rapid Amplification of cDNA Ends
The 5' ends of the A. castellanii and H. vermiformis mtTFB mRNA sequences were confirmed using 5' rapid amplification of cDNA ends (5' RACE). For reverse transcription, 0.5 pmol of reverse primer was annealed to 20 µg of total RNA at 65°C for 5 min, 47°C for 10 min, and room temperature for 10 min. cDNA was synthesized using Superscript II reverse transcriptase (Invitrogen) at 47°C for 45 min following the manufacturer's protocol. The cDNA samples were then treated with ribonuclease H, extracted with phenol, precipitated with ethanol, and resuspended in 10 µl distilled water. The resulting cDNAs were then 3' end tailed with 20 µM deoxyguanosine 5'-triphosphate (dGTP) using terminal deoxynucleotidyltransferase (Invitrogen) according to the manufacturer's instructions. Samples were heated at 90°C for 3 min, and 1.5 µl of sample was used as template for PCR amplification. The first round of amplification was performed with 10 pmol each of the RT-specific primer used for cDNA synthesis and the oAR8 primer under the following cycling parameters: 94°C (4 min); five cycles of 94°C, 50°C, 72°C (30 s each); five cycles of 94°C, 52.5°C, 72°C (30 s each); 25 cycles of 94°C, 55°C, 72°C (30 s each); and 72°C (10 min). A second round of amplification was then performed using 10 pmol each of a nested-specific primer and the oAR6 primer as follows: 3 min at 94°C; 35 cycles of 30 s at 94°C, 30 s at 55°C, 30 s at 72°C; and 10 min at 72°C. PCR products were isolated, cloned, and sequenced as above.

Sequence Analyses
Searches for gene and protein sequences corresponding to mtTFB homologs were carried out in relevant sequence databases (table 2) using TBlastN or BlastP with the various mtTFB sequences as queries. PSI-Blast was also performed using confirmed mtTFB homologs. Translated amino acid sequences of selected genes were aligned with ClustalX 1.82 (Chenna et al. 2003) applying default alignment parameters. Sequence alignments were then adjusted manually using BioEdit (Hall 1999). Preliminary sequence data for Trichomonas vaginalis were obtained from The Institute for Genomic Research (http://www.tigr.org). Sequence data for Thalassiosira pseudonana and Ciona intestinalis were generated by the U.S. Department of Energy Joint Genome Institute (http://www.jgi.doe.gov/). Intron data for h-mtTFB1 and h-mtTFB2 genes were extracted from GenBank entries NP_057104 and NP_071761, respectively, via the National Center for Biotechnology Information Entrez.

View this table: [in this window] [in a new window]   Table 2 Genomic Data Searched for mtTFB Homologs

 
Phylogenetic Analysis
Representative sequences were chosen for various analyses, and sequence alignments were constructed and trimmed manually to remove regions of ambiguity. Initially, trees were generated using Tree-Puzzle with the estimated values then employed to generate neighbor-joining (NJ) and maximum-likelihood (ML) bootstrap values (via Mega3 and PROML, respectively) using a + invariable model. Sequences comprising the alignments are listed in table 3.

View this table: [in this window] [in a new window]   Table 3 Sequences Used in the Generation of Alignments and Phylogenetic Trees

 

	Results

TOP Abstract Introduction Materials and Methods Results Discussion Conclusions Supplementary Material Acknowledgements References

 
Identification of TFB Homologs
ESTs corresponding to the nearly complete sequence of a putative mtTFB were identified in data obtained from the A. castellanii project of the Protist EST Program (http://amoebidia.bcm.umontreal.ca/public/pepdb/agrm.php, ESTs ACE00004873 and ACE00004967). Using these ESTs as query for Blast analysis, a putative TFB homolog was identified in D. discoideum (accession number DDB0186850). An initial alignment of these putative mtTFB sequences with the known mtTFB sequences of fungi and metazoans allowed the identification of conserved sequence motifs and the design of degenerate primers for PCR. A portion of the putative mtTFB from H. vermiformis was successfully amplified using the degenerate primers. The complete genomic sequences were then obtained for both A. castellanii and H. vermiformis using a combination of genomic PCR and RS-PCR, with the assigned methionine initiation codon confirmed by 5' RACE.

Databases of several completely sequenced eukaryotic genomes (listed in table 2), in addition to available ongoing genome and EST projects, were then searched for the presence of mtTFB homologs using BlastX and TBlastN with a variety of mtTFB sequences as queries. Identification of mtTFB homologs through Blast analysis was unsuccessful for most eukaryotes outside of the opisthokonts and amoebozoans (a positive result is defined as one in which the closest reciprocal Blast hit was not a nuclear DMT). However, with the putative A. castellanii mtTFB sequence as query, a mtTFB homolog was identified in each of three sequenced trypanosomatid genomes (GenBank accession numbers AAM97535, XP_843186, and EAN85933).

Sequence Alignment
The putative mtTFB sequences identified here were aligned with known opisthokont mtTFB homologs and representative DMT sequences from the domains Bacteria, Archaea, and Eucarya (fig. 1). Compared to its A. castellanii ortholog, the putative H. vermiformis mtTFB sequence contains an insertion of 29 amino acids (box A, fig. 1), which 5' RACE analysis confirmed was not an intron. As mapped onto the three-dimensional structure of the E. coli KsgA protein (O'Farrell, Scarsdale, and Rife 2004), this insertion is predicted to localize on the exterior of the protein where it is not expected to cause a major structural perturbation. As annotated, the putative D. discoideum mtTFB homolog contains a carboxy-terminal extension of 168 amino acids compared to its A. castellanii counterpart (data not shown).

View larger version (99K): [in this window] [in a new window]   FIG. 1.— The most highly conserved portions of an alignment showing representative mtTFB and DMT homologs used in this study (full alignment available as supplementary fig. 1, Supplementary Material online). Shaded in gray and labeled below the alignment are eight sequence motifs common to methyltransferases, with white letters on a black background indicating residues that make direct contact with the S-adenosylmethionine moiety in ErmC', a 23S rRNA methyltransferase (Bussiere et al. 1998). Secondary structure elements from sc-mtTFB (Schubot et al. 2001) and Escherichia coli KsgA (as aligned to mtTFBs in O'Farrell, Scarsdale, and Rife 2004) are indicated above the alignment (sc-mtTFB at the top), with black arrows indicating ß-sheets and gray cylinders denoting -helices. Sequence insertions of interest are boxed and labeled as follows: A is the Hartmannella vermiformis–specific insertion confirmed by 5' RACE, B and C are putative mtTFB insertions, and D is the yeast-specific mtTFB insertion. Conserved amino acid positions in insertions B and C are indicated in bold letters. Overall positions of sequence conservation as identified by ClustalX (Chenna et al. 2003) are indicated below the alignment. An asterisk (*) indicates a single residue fully conserved in all sequences, whereas a colon (:) or period (.) indicates a strongly or weakly conserved position, respectively. The underlined number on the right-hand side of the alignment indicates the overall position in the alignment with the numbers below showing the amino acid position for each particular protein. Sequences used and corresponding accession numbers are listed in table 3.

 
Further inspection of the alignment in figure 1 revealed putative sequence insertions (boxes B and C) shared by the opisthokont mtTFBs and the mtTFB homologs of amoebozoans and trypanosomatids, compared to the DMTs from bacterial, archaeal, and eukaryotic representatives. In the trypanosomatid versions compared to all other sequences, insertion B is preceded by an additional 80–99 amino acids (supplementary fig. 1, Supplementary Material online). In pairwise BlastP comparisons of the three sequences, this additional stretch exhibits amino acid sequence identity ranging from 31% to 43% and similarity from 50% to 56%. Intriguingly, insertions B and C are predicted to occur in external loops, in the same region of the three-dimensional structure according to available structural data (Schubot et al. 2001; O'Farrell, Scarsdale, and Rife 2004).

Analysis of Introns in mtTFB Genes
An analysis of introns from the putative amoebozoan mtTFB genes shows that A. castellanii and H. vermiformis each has seven introns, whereas the D. discoideum gene has none. The introns are all small, with average sizes of 99 and 63 bp for A. castellanii and H. vermiformis, respectively. Three introns are present at exactly the same position in the A. castellanii and H. vermiformis genes (fig. 2A). Furthermore, when one compares the amoebozoan mtTFB introns to the introns in h-mtTFB1 and h-mtTFB2, there are five positions at which introns are located in one or both of the amoebozoan sequences and at exactly the same position in one or both of the h-mtTFB sequences (fig. 2B). This conservation includes two intron sites that are shared by all four sequences. The h-TFB1 and h-TFB2 genes contain six and seven introns, with average sizes of 9,218 and 2,306 bp, respectively. Not unexpectedly, the trypanosomatids, whose genes are intron poor (Mair et al. 2000; Ivens et al. 2005), have mtTFB homologs that are predicted to contain no introns.

View larger version (12K): [in this window] [in a new window]   FIG. 2.— (A) Analysis of introns in mtTFB genes from three members of Amoebozoa. The solid horizontal line represents the 945 bp of the intron-less mtTFB gene from Dictyostelium discoideum upstream of a nonconserved 3' extension of 510 bp. Vertical bars above and below this line represent Hartmannella vermiformis and Acanthamoeba castellanii intron positions, respectively, with the height of the bars proportional to intron size (numbers indicate intron length in base pairs). Intron phases are depicted by the shading of the bars: white (phase 0), gray (phase 1), and black (phase 2). (B) Intron positions in the Homo sapiens mtTFB1 and mtTFB2 genes, represented by arrowheads (above and below, respectively) on the horizontal line representing the intron-less mtTFB gene from D. discoideum. Intron length is indicated (in base pairs), and intron phases are depicted by the shading of the arrowheads, as in (A). Intron positions conserved between at least one amoebozoan mtTFB and one h-mtTFB are joined by a dotted vertical line.

 
Phylogenetic Analysis
Genes encoding proteins targeted to mitochondria are often of endosymbiont (eubacterial) origin; for this reason, we decided to investigate the likely evolutionary origins of the mtTFB genes. A phylogenetic analysis was performed using representative DMTs from bacteria, archaeons, and eukaryotes in addition to representative mtTFB homologs (fig. 3). In this analysis, shown rooted with the DMT of Pyrococcus abyssi (an archaeon), the eukaryotic DMTs form a strongly supported group (with quartet-puzzling (QP)/NJ/ML bootstrap values of 78/99/96) to the exclusion of mtTFBs, which originate from within the bacterial (+the archaeon Pyrobaculum aerophilum) DMT sequences (80/63/69), where they form a distinct group with the -proteobacterium A. tumefaciens (66/79/63).

View larger version (19K): [in this window] [in a new window]   FIG. 3.— Phylogenetic analysis of representative mtTFB orthologs and a variety of DMT sequences. The QP tree generated using Tree-Puzzle 5.2 is shown rooted with the Pyrococcus abyssi sequence, with branches having QP reliability under 50 collapsed. Support values relevant to the analysis are mapped onto corresponding nodes (QP reliability/NJ bootstrap/ML bootstrap, QP/NJ/ML). Trees were generated using a ClustalX alignment of 153 amino acid positions with ambiguous regions removed (supplementary fig. 2, Supplementary Material online). The NJ and ML values (generated using Mega3 and PROML, respectively) used a + invariable model with parameters = 2.45, eight rate categories, and invariable proportion of 0.01 as estimated by Tree-Puzzle. QP support values were estimated from 1,000 QP steps, while ML and NJ support values were estimated from 100 and 500 bootstrap replicates, respectively. The scale bar represents 0.1 substitutions per site. Sequences used and corresponding accession numbers are listed in table 3. The following abbreviations are used: S. pombe (Schizosaccharomyces pombe), S. cerevisiae (Saccharomyces cerevisiae), and D. melanogaster (Drosophila melanogaster).

 
The long branches represented by the fungal mtTFB and metazoan mtTFB2 genes are indicative of divergent sequences, which may affect statistical support and tree topology through a long-branch attraction (LBA) artifact (Felsenstein 1978). Because of concerns about LBA and in view of the fact that the roles of mtTFB2 and fungal mtTFB as transcription factors had previously been established (Lisowsky and Michaelis 1988; Falkenberg et al. 2002), we removed the fungal sequences in subsequent phylogenetic analyses.

When the analysis was repeated with mtTFB2 and fungal mtTFB sequences were removed, and with additional mtTFB and DMT representative sequences included, the eukaryotic DMT grouping was more strongly supported (89/99/93), as was the affinity of mtTFB sequences specifically with the -proteobacterium A. tumefaciens (89/73/63), though the overall support grouping the mtTFBs and the bacterial DMTs (+the archaeon P. aerophilum) was slightly lower (66/68/61) (fig. 4). Having clearly established that mtTFB homologs are not derived from the eukaryotic DMTs, we examined more closely their bacterial origins. Even with a more comprehensive and diverse range of bacterial DMT representatives including a range of -proteobacteria, the resulting tree (fig. 5) still supports an origin of mtTFB from within -proteobacteria (QP/NJ/ML bootstrap values of 82/87/60).

View larger version (26K): [in this window] [in a new window]   FIG. 4.— Phylogenetic analysis of representative mtTFB orthologs (with long-branching representatives from fig. 3 removed) and a variety of DMT sequences. Trees were generated as described in figure 3 using a ClustalX alignment of 157 amino acid positions with ambiguous regions removed (supplementary fig. 3, Supplementary Material online). The NJ and ML values (generated using Mega3 and PROML, respectively) used a + invariable model with parameters = 2.31, eight rate categories, and invariable proportion of 0.04 as estimated by Tree-Puzzle. QP support values were estimated from 1,000 QP steps, while ML and NJ support values were estimated from 100 and 500 bootstrap replicates, respectively. The scale bar represents 0.1 substitutions per site. Sequences used and corresponding accession numbers are listed in table 3. Trypanosoma brucei and Leishmania major are abbreviated as T. brucei and L. major, respectively.

 

View larger version (30K): [in this window] [in a new window]   FIG. 5.— Phylogenetic analysis of representative mtTFB (Tc) and DMT sequences from bacteria (-proteobacteria in bold). Trees were generated as described in figure 3 using a ClustalX alignment of 157 amino acid positions with ambiguous regions removed (supplementary fig. 4, Supplementary Material online). The NJ and ML values (generated using Mega3 and PROML, respectively) used a + invariable model with parameters = 2.02, eight rate categories, and invariable proportion of 0.05 as estimated by Tree-Puzzle. QP support values were estimated from 1,000 QP steps, while ML and NJ support values were estimated from 100 and 500 bootstrap replicates, respectively. The scale bar represents 0.1 substitutions per site. Sequences used and corresponding accession numbers are listed in table 3.

 

	Discussion

TOP Abstract Introduction Materials and Methods Results Discussion Conclusions Supplementary Material Acknowledgements References

 
Phylogenetic analysis supports the view that mtTFB arose from within -proteobacteria, in turn strongly indicating an origin of mtTFB from a DMT gene present in the proto-mitochondrial endosymbiont, rather than from a nuclear DMT gene of the eukaryotic host. In addition to the phylogenetic evidence, conservation of five intron positions further supports the shared relationship between opisthokont and amoebozoan mtTFBs. However, the sparse distribution of mtTFB homologs within eukaryotes is notable and intriguing with regard to regulation of mitochondrial transcription.

Transcription Factor Activity
As outlined in the Introduction, initiation of mitochondrial transcription in general and the role of mtTFBs specifically have been studied in three model systems (human, Saccharomyces cerevisiae, and Drosophila). However, no consensus model has emerged describing the precise role of mtTFB and the particulars of its transcription factor activity. At some point during its evolution, the mitochondrial DMT evidently acquired a secondary function (that of a transcription factor) in addition to its original methyltransferase activity. Given that both fungal and metazoan mtTFB homologs exhibit transcription factor activity, it is likely that this function was acquired prior to the fungal-animal divergence. However, the exact timing of this novel functional acquisition is difficult to determine due to the sparse phylogenetic distribution of mtTFB homologs and lack of characterized mtTFs outside of the opisthokonts.

In humans, the transcription factor activity of mtTFB1 is independent of its ability to bind S-adenosylmethionine and dimethylate SSU rRNA in vitro (McCulloch and Shadel 2003). While DMT activity has been well characterized and the structures of E. coli KsgA and sc-mtTFB were solved (Schubot et al. 2001; O'Farrell, Scarsdale, and Rife 2004), it is not known how the mtTFB mediates transcription and which portions of the protein are involved. In yeast, an insertion in sc-mtTFB (box D in fig. 1) has been proposed to be involved in its transcription factor activity (Schubot et al. 2001). However, while the yeast insertion may affect mtTFB transcription factor activity, it is yeast-specific and thus cannot be the sole determinant. It is plausible that the putative mtTFB sequence insertions found in all known mtTFBs and the mtTFB homologs identified here may be important in this regard, in particular box C where four of the six residues are conserved. In the three-dimensional structure of E. coli KsgA (O'Farrell, Scarsdale, and Rife 2004), these insertions are adjacent to one another at the surface of the protein such that they could be involved in protein-protein interactions.

Distribution of mtTFB Homologs
Due to the fact that ongoing genome projects and EST databases are incomplete, absence of mtTFB homologs in these databases is not necessarily definitive. Nevertheless, and somewhat surprisingly, when completely sequenced genomes are examined, mtTFB homologs appear to be restricted to opisthokonts (metazoan animals + fungi), amoebozoans, and trypanosomatids. Finding mtTFB homologs within Amoebozoa is not unexpected, given the view that this clade shares a common ancestry with the opisthokonts to the exclusion of all other eukaryotic supergroups (Fahrni et al. 2003). The trypanosomatids, however, belong to an unrelated eukaryotic clade, so it is surprising that this is the only other lineage in which mtTFB homologs have been identified. Barring the possibility that mtTFB homologs have diverged beyond recognition (discussed below) in other eukaryotic taxa, this punctate distribution might be explained in a number of ways.

Considering the -proteobacterial ancestry of the mtTFB, the originating gene was most likely transferred from the mitochondrial endosymbiont genome to the nucleus. This transfer could have occurred once in the ancestor of extant eukaryotes, with subsequent losses of the nucleus-encoded copy in lineages lacking an mtTFB homolog. A more complex scenario would involve multiple endosymbiont-to-nucleus transfers in some lineages (i.e., opisthokont/amoebozoa and trypanosomatids), with other lineages either losing the ancestral DMT gene directly from the mitochondrial genome or from the nuclear genome after transfer.

A third possibility is that of a lateral gene transfer from one lineage to another, most likely opisthokont/amoebozoan trypanosomatid, given the apparent distribution of homologs. Such a scenario would obviously reduce the number of mitochondrion-to-nuclear transfers and subsequent losses required to explain the observed phylogenetic distribution of mtTFB. Furthermore, this proposal would be consistent with recent views on the root of eukaryotes, wherein opisthokonts and amoebozoans are combined in an assemblage referred to as "unikonts" (Keeling et al. 2005; Richards and Cavalier-Smith 2005). In this sense, the origin of a mtTFB in mitochondrial transcription might be specific to the unikonts as opposed to the rest of the eukaryotes, where a mtTF may not be required. The latter situation would mimic the activity of T7-phage RNAP, which is known to function without any accessory TF proteins. However, current data neither support nor refute the possibility of lateral gene transfers.

Dimethylation of Mitochondrial SSU rRNA
If one accepts that homologs of mtTFB are not, in fact, widely distributed throughout the eukaryotic radiation, such a restricted occurrence raises two questions: (1) how is mitochondrial transcription regulated in the absence of a mtTFB? and (2) how are mitochondrial SSU rRNAs dimethylated, if in fact they are? Considering the second question together with the possibility that mtTFB homologs may not be easily identifiable with current methods due to sequence divergence, we searched the literature for information regarding dimethylation of mitochondrial SSU rRNA corresponding to E. coli SSU (16S) rRNA residues A1518 and A1519.

Little is actually known about the state of SSU rRNA modifications in mitochondria due to the technical challenges involved in such analyses. However, dimethylation at these sites (or lack thereof) has been inferred through direct chemical sequencing experiments carried out on the mitochondrial SSU rRNA in a few mitochondrial systems. Thus, in mammals and the amoebozoan A. castellanii, both of which contain mtTFB homologs, dimethylation of mitochondrial SSU rRNA has been inferred (Baer and Dubin 1980; Lonergan and Gray 1994). However, in the ciliate Tetrahymena pyriformis (a member of the Chromalveolata), SSU rRNA dimethylation evidently does not occur (Schnare et al. 1986). This observation is consistent with the lack of an identifiable mtTFB homolog in the genomes of the close relative Tetrahymena thermophila and several other chromalveolates (table 2). A similar situation is seen in the green alga Polytomella parva, where the mitochondrial SSU rRNA is not dimethylated (Fan, Schnare, and Lee 2003), again consistent with the lack of an identifiable mtTFB homolog in the completely sequenced genome of the red alga Cyanidioschyzon merolae (a member of the same supergroup, Plantae, as P. parva) and the nearly complete genome sequence of the closely related green alga Chlamydomonas reinhardtii.

The situation in land plants differs from that in the red and green algae. Dimethylation of the mitochondrial SSU rRNA has been inferred in wheat, Triticum aestivum (Schnare and Gray 1982), although no mtTFB homolog has been identified in the completely sequenced genome of Arabidopsis thaliana. There are, however, three homologs in the A. thaliana genome exhibiting strong hits to DMTs. These sequences include a plastid DMT that has been previously identified (GenBank accession number AAC09322) (Tokuhisa et al. 1998) and two gene homologs that clearly encode eukaryotic DMTs (Entrez genes At5g66360 and At2g47420). The At5g66360 gene gives rise to two splice variants (NP_975003 and NP_201437), both of which are putatively targeted to mitochondria (based on a score of 0.671 using the predictive algorithm TargetP (http://www.cbs.dtu.dk/services/TargetP/). A similar eukaryotic DMT duplication is also evident in the Oryza sativa (rice) and Populus trichocarpa (poplar) genomes. Thus, it seems likely that in angiosperms, duplication and divergence of the eukaryotic DMT have provided a copy that functions as the mitochondrial DMT.

The possibility remains that some of these lineages possess a mtTFB homolog that has lost DMT activity and which instead specializes solely as a transcription factor. Such a situation, in which functional constraints are reduced, could potentially allow the sequence to evolve to such an extent that it is no longer recognizable on the basis of sequence comparisons. This scenario would be reminiscent of the case of yeast sc-mtTFB, which lacks DMT activity and is considerably more divergent in sequence than other mtTFB homologs. Similarly, in Metazoa, the divergent h-mtTFB2 duplicate is 10 times more effective as a transcription factor than h-mtTFB1 (Falkenberg et al. 2002). However, it is not known whether mtTFB2 retains any DMT activity.

It is possible that in other lineages, duplication and divergence of a eukaryotic nucleus-encoded DMT have also served to provide a functional mitochondrial DMT. In such a situation, the resulting mitochondrion-targeted DMT would not necessarily be expected to act as a transcription factor in mtDNA replication. On the other hand, such a duplication might allow a separate, endosymbiont-derived mtTFB to specialize as a transcription factor, in which case it might well diverge to such an extent that it is no longer recognizable as a typical mtTFB. Another possibility is that a nucleus-encoded plastid DMT could also function as a mitochondrial DMT. However, while the products of mitochondrial and chloroplast genes can be shared in A. thaliana (MacKenzie 2005), there is currently no evidence to suggest that this occurs in the case of DMTs.

	Conclusions

TOP Abstract Introduction Materials and Methods Results Discussion Conclusions Supplementary Material Acknowledgements References

 
The mtTFB is an interesting example of a mitochondrial protein (a methyltransferase) acquiring a second, apparently unrelated function (that of transcription factor). In phylogenetic analyses, the novel eukaryotic mtTFB homologs identified in this study group together with the yeast and metazoan mtTFBs, and they all appear to have originated from the endosymbiont genome. However, the eukaryotic distribution (or lack thereof) of mtTFB homologs makes it difficult to infer the evolutionary history within the eukaryotes, in particular with regard to the origins of the transcription factor activity.

The mechanism of mitochondrial transcription activation is yet to be fully resolved, in particular the role of mtTFB. Thus, an examination of transcription in the mitochondria of earlier diverging eukaryotes, such as members of Amoebozoa, may provide insight into an ancestral transcription mechanism and help us to elucidate common features. This problem is compounded by the possible existence of mtTFB homologs that cannot be readily identified due to sequence divergence. In this context, development of in vitro mitochondrial transcription systems and functional tests for mtTF activity take on added significance. It will also be of interest to examine mitochondrial transcription in organisms that appear to lack mtTFB.

The biochemistry of transcription in mitochondria is likely to be as variable as the genomes on which they act. This is evidenced by the differences in mtDNA transcription between human, yeast, and Drosophila with regard to mtTFA, promoter recognition, and protein interactions. Thus, biochemical studies on one or even a few "model" systems will be insufficient to enable us to understand the full range of biochemical properties and the evolution of the mitochondrial transcription system. In this regard, a combination of bioinformatic and biochemical approaches to study a variety of eukaryotic systems will likely play an increasingly important role in studying mitochondrial transcription.

	Supplementary Material

TOP Abstract Introduction Materials and Methods Results Discussion Conclusions Supplementary Material Acknowledgements References

 
Supplementary figs. 1–4 are available at Molecular Biology and Evolution online (http://www.mbe.oxfordjournals.org/).

	Acknowledgements

TOP Abstract Introduction Materials and Methods Results Discussion Conclusions Supplementary Material Acknowledgements References

 
We are grateful to A. J. Lohan and C. E. Bullerwell for donating nucleic acid samples used in this study. We thank R. W. Watkins for help with phylogenetic analysis and M. Dutlek for performing DNA sequencing. Preliminary sequence data for T. vaginalis were obtained from The Institute for Genomic Research Web site (http://www.tigr.org). Sequencing of the T. vaginalis genome was funded by the National Institute of Allergy and Infectious Diseases. Sequence data for T. pseudonana and C. intestinalis were produced by the U.S. Department of Energy Joint Genome Institute (http://www.jgi.doe.gov/). This work was supported by funding to M.W.G. from the Canadian Institutes of Health Research (operating grant MOP-4355). EST data were generated under the auspices of the Protist EST Program, with funding through Genome Canada/Genome Atlantic and the Atlantic Innovation Fund. M.W.G. is pleased to acknowledge salary support from the Canada Research Chairs Program and the Canadian Institute for Advanced Research.

	Footnotes

 
Martin Embley, Associate Editor

	References

TOP Abstract Introduction Materials and Methods Results Discussion Conclusions Supplementary Material Acknowledgements References

 

Baer, R., and D. T. Dubin. 1980. The 3'-terminal sequence of the small subunit ribosomal RNA from hamster mitochondria. Nucleic Acids Res. 8:4927–4941.[Abstract/Free Full Text]

Burger, G., M. W. Gray, and B. F. Lang. 2003. Mitochondrial genomes: anything goes. Trends Genet. 19:709–716.[CrossRef][Web of Science][Medline]

Bussiere, D. E., S. W. Muchmore, C. G. Dealwis, G. Schluckebier, V. L. Nienaber, R. P. Edalji, K. A. Walter, U. S. Ladror, T. F. Holzman, and C. Abad-Zapatero. 1998. Crystal structure of ErmC', an rRNA methyltransferase which mediates antibiotic resistance in bacteria. Biochemistry 37:7103–7112.[CrossRef][Medline]

Cermakian, N., T. M. Ikeda, R. Cedergren, and M. W. Gray. 1996. Sequences homologous to yeast mitochondrial and bacteriophage T3 and T7 RNA polymerases are widespread throughout the eukaryotic lineage. Nucleic Acids Res. 24:648–654.[Abstract/Free Full Text]

Cheetham, G. M., and T. A. Steitz. 2000. Insights into transcription: structure and function of single-subunit DNA-dependent RNA polymerases. Curr. Opin. Struct. Biol. 10:117–123.[CrossRef][Web of Science][Medline]

Chenna, R., H. Sugawara, T. Koike, R. Lopez, T. J. Gibson, D. G. Higgins, and J. D. Thompson. 2003. Multiple sequence alignment with the Clustal series of programs. Nucleic Acids Res. 31:3497–3500.[Abstract/Free Full Text]

Fahrni, J. F., I. Bolivar, C. Berney, E. Nassonova, A. Smirnov, and J. Pawlowski. 2003. Phylogeny of lobose amoebae based on actin and small-subunit ribosomal RNA genes. J. Mol. Evol. 20:1881–1886.

Falkenberg, M., M. Gaspari, A. Rantanen, A. Trifunovic, N. G. Larsson, and C. M. Gustafsson. 2002. Mitochondrial transcription factors B1 and B2 activate transcription of human mtDNA. Nat. Genet. 31:289–294.[CrossRef][Web of Science][Medline]

Fan, J., M. N. Schnare, and R. W. Lee. 2003. Characterization of fragmented mitochondrial ribosomal RNAs of the colorless green alga Polytomella parva. Nucleic Acids Res. 31:769–778.[Abstract/Free Full Text]

Felsenstein, J. 1978. Cases in which parsimony or compatibility methods will be positively misleading. Syst. Zool. 27:401–410.[CrossRef][Web of Science]

Grams, J., J. C. Morris, M. E. Drew, Z. Wang, P. T. Englund, and S. L. Hajduk. 2002. A trypanosome mitochondrial RNA polymerase is required for transcription and replication. J. Biol. Chem. 277:16952–16959.[Abstract/Free Full Text]

Gray, M. W., G. Burger, and B. F. Lang. 1999. Mitochondrial evolution. Science 283:1476–1481.[Abstract/Free Full Text]

Hall, T. A. 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp. Ser. 41:95–98.

Hedtke, B., T. Börner, and A. Weihe. 1997. Mitochondrial and chloroplast phage-type RNA polymerases in Arabidopsis. Science 277:809–811.[Abstract/Free Full Text]

Helser, T. L., J. E. Davies, and J. E. Dahlberg. 1971. Change in methylation of 16S ribosomal RNA associated with mutation to kasugamycin resistance in Escherichia coli. Nat. New Biol. 233:12–14.[Web of Science][Medline]

Ivens, A. C., C. S. Peacock, E. A. Worthey et al. (104 co-authors). 2005. The genome of the kinetoplastid parasite, Leishmania major. Science 309:436–442.[Abstract/Free Full Text]

Jang, S. H., and J. A. Jaehning. 1991. The yeast mitochondrial RNA polymerase specificity factor, MTF1, is similar to bacterial sigma factors. J. Biol. Chem. 266:22671–22677.[Abstract/Free Full Text]

Keeling, P. J., G. Burger, D. G. Durnford, B. F. Lang, R. W. Lee, R. E. Pearlman, A. J. Roger, and M. W. Gray. 2005. The tree of eukaryotes. Trends Ecol. Evol. 20:670–676.

Klootwijk, J., I. Klein, and L. A. Grivell. 1975. Minimal post-transcriptional modification of yeast mitochondrial ribosomal RNA. J. Mol. Biol. 97:337–350.[CrossRef][Web of Science][Medline]

Lisowsky, T., and G. Michaelis. 1988. A nuclear gene essential for mitochondrial replication suppresses a defect of mitochondrial transcription in Saccharomyces cerevisiae. Mol. Gen. Genet. 214:218–223.[CrossRef][Web of Science][Medline]

Lonergan, K. M., and M. W. Gray. 1994. The ribosomal RNA gene region in Acanthamoeba castellanii mitochondrial DNA. A case of evolutionary transfer of introns between mitochondria and plastids? J. Mol. Biol. 239:476–499.[CrossRef][Web of Science][Medline]

Mackenzie, S. A. 2005. Plant organellar protein targeting: a traffic plan still under construction. Trends Cell Biol. 15:548–554.[CrossRef][Web of Science][Medline]

Mair, G., H. Shi, H. Li et al. (14 co-authors). 2000. A new twist in trypanosome RNA metabolism: cis-splicing of pre-mRNA. RNA 6:163–169.[Abstract]

Masters, B. S., L. L. Stohl, and D. A. Clayton. 1987. Yeast mitochondrial RNA polymerase is homologous to those encoded by bacteriophages T3 and T7. Cell 51:89–99.[CrossRef][Web of Science][Medline]

McCulloch, V., B. L. Seidel-Rogol, and G. S. Shadel. 2002. A human mitochondrial transcription factor is related to RNA adenine methyltransferases and binds S-adenosylmethionine. Mol. Cell. Biol. 22:1116–1125.[Abstract/Free Full Text]

McCulloch, V., and G. S. Shadel. 2003. Human mitochondrial transcription factor B1 interacts with the C-terminal activation region of h-mtTFA and stimulates transcription independently of its RNA methyltransferase activity. Mol. Cell. Biol. 23:5816–5824.[Abstract/Free Full Text]

O'Farrell, H. C., J. N. Scarsdale, and J. P. Rife. 2004. Crystal structure of KsgA, a universally conserved rRNA adenine dimethyltransferase in Escherichia coli. J. Mol. Biol. 339:337–353.[CrossRef][Web of Science][Medline]

Richards, T. A., and T. Cavalier-Smith. 2005. Myosin domain evolution and the primary divergence of eukaryotes. Nature 436:1113–1118.[CrossRef][Medline]

Sarkar, G., R. T. Turner, and M. E. Bolander. 1993. Restriction-site PCR: a direct method of unknown sequence retrieval adjacent to a known locus using universal primers. PCR Methods Appl. 2:318–322.[Medline]

Schnare, M. N., and M. W. Gray. 1982. 3'-Terminal sequence of wheat mitochondrial 18S ribosomal RNA: further evidence of a eubacterial evolutionary origin. Nucleic Acids Res. 10:3921–3932.[Abstract/Free Full Text]

Schnare, M. N., T. Y. Heinonen, P. G. Young, and M. W. Gray. 1986. A discontinuous small subunit ribosomal RNA in Tetrahymena pyriformis mitochondria. J. Biol. Chem. 261:5187–5193.[Abstract/Free Full Text]

Schubot, F. D., C. J. Chen, J. P. Rose, T. A. Dailey, H. A. Dailey, and B. C. Wang. 2001. Crystal structure of the transcription factor sc-mtTFB offers insights into mitochondrial transcription. Protein Sci. 10:1980–1988.[CrossRef][Web of Science][Medline]

Seidel-Rogol, B. L., V. McCulloch, and G. S. Shadel. 2003. Human mitochondrial transcription factor B1 methylates ribosomal RNA at a conserved stem-loop. Nat. Genet. 33:23–24.[CrossRef][Web of Science][Medline]

Simpson, A. G, and A. J. Roger. 2004. The real ‘kingdoms’ of eukaryotes. Curr. Biol. 14:R693–R696.[CrossRef][Web of Science][Medline]

Tokuhisa, J. G., P. Vijayan, K. A. Feldmann, and J. A. Browse. 1998. Chloroplast development at low temperatures requires a homolog of DIM1, a yeast gene encoding the 18S rRNA dimethylase. Plant Cell 10:699–711.[Abstract/Free Full Text]

Weber, K. L., M. E. Bolander, and G. Sarkar. 1998. Rapid acquisition of unknown DNA sequence adjacent to a known segment by multiplex restriction site PCR. Biotechniques 25:415–419.[Web of Science][Medline]

Xu, B., and D. A. Clayton. 1992. Assignment of a yeast protein necessary for mitochondrial transcription initiation. Nucleic Acids Res. 20:1053–1059.[Abstract/Free Full Text]

Accepted for publication March 6, 2006.

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