Functional conservation of yeast mtTFB despite extensive sequence divergence

Abstract

Transcription of mtDNA in the yeast S. cerevisiae depends on recognition of a consensus nonanucleotide promoter sequence by mtRNA polymerase acting with a 40-kDa dissociable factor known as mtTFB or Mtflp. mtTFB has been cloned and characterized in S. cerevisiae, but has not been studied in similar detail in any other organism. Although it is known that mitochondrial transcription in the dairy yeast, Kluyveromyces lactis, initiates within the same consensus promoter sequence used in S. cerevisiae, no previous studies have focused on the proteins involved in transcription initiation in K. lactis. In this article, we report the cloning of mtTFB from K. lactis and from a yeast more closely related to S. cerevisiae, S. kluyveri. Both novel mtTFB genes were able to substitute for the MTF1 gene in S. cerevisiae. Both proteins purified following expression in E. coli were able to support specific transcription initiation in vitro with the S. cerevisiae mtRNA polymerase. The S. kluyveri and K. lactis mtTFB proteins share only 56% and 40% identity with S. cerevisiae mtTFB, respectively. Alignments of the three mtTFB sequences did not reveal any regions larger than 30 amino acids with greater than 60% amino acid identity. In particular, regions proposed to show sequence similarity to bacterial sigma factors were not more highly conserved than other regions of the mtTFB proteins. All three yeast mtTFB genes lack conventional amino-terminal mitochondrial targeting sequences, suggesting that all three proteins may be imported into mitochondria by the same unusual mechanism reported for S. cerevisiae mtTFB.

FIG. 1

Putative sk-mtTFB and kl-mtTFB genes occur as single-copy genes. Autoradiograms are shown of DNA blots in which 10 μg of genomic yeast DNA was hybridized with homologous labeled probes from putative mtTFB genes as described in Materials and Methods. The positions of mobility markers in a commercial 1-kb DNA ladder (Life Technologies) are noted on each gel.

FIG. 2

Alignment of three yeast mtTFB protein sequences. Sequences were aligned using the program Clustal (PCGene). Residues marked by asterisks (*) are identical in all three sequences. Residues marked by periods (.) either match at two of three positions or have chemically similar residues at the indicated position in all three proteins, as defined in Fig. 5. The symbols above the sequence for sc-mtTFB show the effects of altering the indicated amino acids in the sc-mtTFB sequence (30), as noted in the figure and discussed in the text. Genbank accession numbers for the sequences are “U81619” and “U81620” for S. kluyveri and K. lactis mtTFB, respectively.

FIG. 3

Recombinant mtTFB proteins from heterologous yeasts can replace sc-mtTFB in in vitro transcription by sc-mtRNA polymerase from a canonical nonanucleotide promoter. In vitro transcription experiments were performed as described in Materials and Methods using sc-mtRNA polymerase alone (lane 2) or mtRNA polymerase plus the indicated amount of recombinant mtTFB proteins expressed from genes obtained from S. cerevisiae (sc), S. kluyveri (sk), or K. lactis (kl). Lane 1 shows a control reaction from which mtRNA polymerase was omitted. In vitro transcripts were fractionated by electrophoresis on a polyacrylamide-urea gel and detected by Phosphorimager analysis. The positions of labeled MspI fragments of pUC18 DNA as gel mobility markers are indicated at the left. The labels on the right are discussed in the text.

FIG. 4

Disruption of the S. cerevisiae MTF1 gene and complementation of sc-mtTFB function by S. kluyveri and K. lactis sc-mtTFB homologues in vivo. (A) The S. cerevisiae MTF1 gene (black arrow) was disrupted by insertion of the HIS3 gene (dashed rectangle) into the coding region (between the existing EcoRV and SphI sites in the MTF1 gene). A His⁺ transformant that contained the chromosomal HIS3 disruption and a copy of the MTF1 gene on a plasmid was designated GS113 and used in the plasmid shuffle experiments (see Materials and Methods for details). (B) Rescue of the MTF1 deletion by mtTFB homologues from S. kluveri and K. lactis sc-mtTFB. GS113 was transformed with plasmids containing the TRP1 gene as a marker and either the S. cerevisiae MTF1 gene (sc-mtTFB), the S. kluveri mtTFB gene (sk-mtTFB), the S. kluveri mtTFB gene with a deletion of the promoter and the beginning of the coding region (sk-mtTFBΔP), the K. lactis mtTFB gene (kl-mtTFB), or no mtTFB insert (pRS314). These strains were replica plated onto YPD (dextrose) and YPG (glycerol) medium after growth on 5-FOA-containing medium to force the loss of the S. cerevisiae MTF1 gene. Growth on YPG indicated rescue of wild-type sc-mtTFB function by the introduced mtTFB homologues (sk-mtTFB and kl-mtTFB) as manifested by maintenance of mitochondrial function. The order in which the strains appear on the plates is given in the key at the bottom of the figure.

FIG. 5

The σ factor homologies in mtTFB sequences. A consensus sequence was derived for the three yeast mtTFB sequences (YB) and domains were aligned to σ2.1/2.2, σ2.3/2.4, and σ3 sequence motifs as identified by Lonetto et al. (21). The alignments are positioned to reflect the sequence alignments reported by Jang and Jaehning (17). Amino acids shown in upper case are highly conserved, whereas those shown in lower case are well conserved (two of three for the yeast mtTFB sequences). Note that the underlined gkp in the sequence aligned with σ3 represents an insertion in the sc-mtTFB and sk-mtTFB sequences with respect to the kl-mtTFB sequence. The numerical code used to denote similar amino acids follows that used by Lonetto et al. (21); x denotes any amino acid. The residues are numbered as in the sc-mtTFB and E. coli σ70 sequences. The symbols above the YB sequence refer to the importance of individual residues as determined by point mutagenesis (30), as defined in Fig. 2.