Control of mitochondrial gene expression in the yeast Saccharomyces cerevisiae.

Abstract

Mitochondrial promoters in Saccharomyces cerevisiae contain an identical octanucleotide [TATAAGTA-(+ 1)] sequence present just upstream of the initiation site (at the left end of the arrow). Studies have shown that the transcription rates of mitochondrial genes vary from 7-to 15-fold. The nucleotide at position +2 regulates the efficiency of mitochondrial promoters but does not affect the specificity of initiation. The data presented herein demonstrate that the variable transcription rates of mitochondrial genes are due to different levels of transcriptional initiation. The rate of first phosphodiester bond formation between a purine and a pyrimidine on a weak promoter is much lower than that of purine-purine on a strong promoter. A dinucleotide corresponding to positions +1 and +2 acts in vitro as a primer, bypassing the first phosphodiester bond formation at the initiation site. When these dinucleotides were used to prime transcription, the activities of the strong and weak promoters were found to be identical. In heparinnchallenge experiments, there is no significant effect of dinucleotide on heparin-resistant DNA-RNA polymerase complex formation. These results indicate that the low level of transcription from the weak mitochondrial promoter is due to the slow rate of formation of the first phosphodiester bond.Significant progress has been made toward an understanding of the control of mitochondrial gene expression in the yeast Saccharomyces cerevisiae (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12). The biogenesis of mitochondria is regulated by the coordinated expression of both mitochondrial and nuclear genes (13). The 76-kilobase mitochondrial DNA of yeast encodes a limited number of mitochondrial proteins, two rRNAs, and a set oftRNAs necessary for translation of mitochondrial mRNAs. The remaining mitochondrial proteins including factors required for mitochondrial transcription, replication, and translation are encoded by the nuclear genome and imported into mitochondrial.Mitochondrial RNA (mtRNA) polymerase consists of a core enzyme and a specificity factor, both of which are required for specific transcription of mitochondrial genes (8-10). The mitochondrial promoter consists of an 8-nucleotide sequence [5'-TATAAGTA(+1)-3'] that has been identified in the mitochondrial rRNA, tRNA, and mRNA genes as well as at the origins of replication (2). In vitro transcription studies of variant promoter mutants showed that a short octanucleotide sequence, 5'-TAtAaGtN-3', is the minimum sequence requirement to direct accurate initiation of transcription by mtRNA polymerase (6). The lowercase letters indicate positions at which nucleotide variants are permissible and N designates any nucleotide that is used as an initiating nucleotide.Although mitochondrial genes share an identical promoter sequence, they are transcribed at different rates (5, 12). The promoters can be grouped into two classes: strong promoters and weak promoters. The strong promoters have 7-15 times the activity of the weak promoters. Promoters are ...

Sensitivity of the Yeast Mitochondrial RNA Polymerase to +1 and +2 Initiating Nucleotides

“…Like other RNAPs, the mtRNAP requires higher levels of its initiating (ϩ1 ATP) nucleotide than those used for elongation (10,11,17). We have recently extended this observation to demonstrate a clear relationship between the in vitro kinetics of ATP utilization (K m ATP) and the in vivo regulation of mtRNAP transcription (18).…”

“…To confirm that the high K m values observed are in fact because of a requirement for initiation, we repeated K m ATP experiments on the COX1 (ϩ1 ϩ2 AA) promoter template in the presence or absence of the dinucleotide ApA to eliminate the energy barrier of first bond formation and stabilize the initiation complex (10,11). Fig.…”

Mutations in the Yeast Mitochondrial RNA Polymerase Specificity Factor, Mtf1, Verify an Essential Role in Promoter Utilization

“…Strong promoters like 14 S rRNA and COX2 have a purine at ϩ2, whereas the weak tRNA Cys promoter has a pyrimidine at ϩ2. Biswas (25) has also shown that promoters that are weak due to a ϩ2 pyrimidine can be corrected in vitro by addition of a dinucleotide corresponding to the ϩ1 and ϩ2 nucleotides of the transcript. Weak promoters that vary from the consensus at positions farther upstream cannot be corrected by dinucleotide addition, possibly due to defects in the initial binding of the RNAP.…”

“…Weak promoters that vary from the consensus at positions farther upstream cannot be corrected by dinucleotide addition, possibly due to defects in the initial binding of the RNAP. Biswas (24,25) has speculated that addition of the dinucleotide either bypasses an energy barrier for first bond formation or stabilizes the initiation complex.…”

“…We first asked whether the defect was at the level of first bond formation by testing the response to addition of dinucleotide primers to the transcription reactions. Biswas (24,25) has shown that transcription from weak promoters such as tRNA Cys is significantly improved when a dinucleotide corresponding to the ϩ1 and ϩ2 positions is provided, whereas transcription from promoter variants with sequence alterations toward the 5Ј-end is unaffected by the addition of dinucleotides. Consistent with these observations, we found little or no effect of dinucleotide addition on transcription of the COX2 promoter by either wild type or mutant Mtf1 (Fig.…”