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

Elizabeth A Amiott,Judith A Jaehning

doi:10.1074/jbc.m608638200

Elizabeth A Amiott, Judith A Jaehning

Open Access

https://doi.org/10.1074/jbc.m608638200

Copy DOI

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.

Highlights

Rpo41, creating a functional “holoenzyme” with promoter-specific activity [5]
The mtRNAP Requires High Concentrations of Both the ϩ1 and ϩ2 NTPs—Using purified recombinant mtRNAP subunits and varying concentrations of a single NTP, we determined the concentration of ATP, GTP, CTP, or UTP substrate at which half-maximal enzymatic activity was achieved (Km) on several mitochondrial promoters in vitro
This work demonstrates that the mtRNAP requires high concentrations of the first two NTPs, in particular the ϩ2 NTP, used to initiate RNA synthesis

Summary

Introduction

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]. Like other RNAPs, the mtRNAP requires higher levels of its initiating (ϩ1 ATP) nucleotide than those used for elongation

Results

Conclusion