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

Abstract

Despite a simple consensus sequence, there is considerable variation of promoter strengths, transcription rates, and the kinetics of initiating nucleotide incorporation among the promoters found in the Saccharomyces cerevisiae mitochondrial genome. We asked how changes in the initiating (؉1 and ؉2) nucleotides, conformation of the promoter DNA template, and mutation of the mitochondrial RNA polymerase (mtRNAP) affect the kinetics of nucleotide (NTP) utilization. Using a highly purified in vitro mitochondrial transcription system, we found that 1) the mtRNAP requires the highest concentrations of the ؉1 and ؉2 initiating NTPs, intermediate concentrations of NTPs at positions 5 to 11, and low concentrations of elongating NTPs; 2) the mtRNAP requires a higher concentration of the ؉2 NTP than the ؉1 NTP for initiation; 3) the kinetics of ؉2 NTP utilization are altered by a point mutation in the mtRNAP subunit Mtf1; and 4) a supercoiled or pre-melted promoter DNA template restores normal ؉2 NTP utilization by the Mtf1 mutant. Based on comparisons to the structural and biochemical properties of the bacterial RNAP and the closely related T7 RNAP, we propose that initiating nucleotides, particularly the ؉2 NTP, are required at high concentrations to drive mitochondrial promoter opening or to stabilize a productive open complex.The core subunit of the budding yeast mitochondrial RNA polymerase (mtRNAP), 3 encoded by the nuclear gene RPO41, is a 145-kDa protein that shares significant amino acid similarity with the T7/T3 bacteriophage family of single subunit RNAPs (1, 2). However, T7 RNAP is a single subunit enzyme capable of specific and regulated activity in the absence of any transcription factors (3), but mtRNAPs from all eukaryotes examined to date require one or more auxiliary factors for selective transcription (reviewed in Ref. 4). The accessory factor for the yeast mtRNAP, Mtf1, is a 43-kDa protein that interacts directly with Rpo41, creating a functional "holoenzyme" with promoter-specific activity (5). Following the synthesis of a short RNA chain (ϳ12 nucleotides), Mtf1 is released, and Rpo41 continues elongation of the transcript (6).Recently it has been demonstrated that Rpo41 alone can initiate transcription in a promoter-specific manner in vitro in the absence of Mtf1 (7). However, this Mtf1-independent activity is only possible on supercoiled or pre-melted "bubble" promoter templates in which the promoter opening stages of transcription initiation have been facilitated or bypassed. Mtf1 alters the ratio of full-length to abortive transcripts on nonlinear templates (7) demonstrating that Mtf1 influences both promoter opening and clearance by the mtRNAP. Despite these findings, the mechanistic function of Mtf1 and its homologs in promoter selective transcription is still not well understood.The yeast mtRNAP mediates gene expression in the mitochondrial compartment by binding and initiating transcription at a collection of short, nonanucleotide (consensus ATATA-AGTA) promoters encompassing the transcriptiona...

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Regulation of Pyrimidine Biosynthetic Gene Expression in Bacteria: Repression without Repressors

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“…Apparently, after formation of the first internucleotide bond, the dinucleotide product stabilizes the transcription initiation complex. Lower concentrations of NTP substrates are required for transcript extension beyond position ϩ2, though the relaxation of the requirement for high NTP concentrations may occur gradually until promoter clearance (2). Based on these observations and the fact that the ϩ2 nucleotide in pyrC C7 transcripts is a C, it appears necessary to make a final modification to the model for pyrC regulation.…”

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