A Promoter Recognition Mechanism Common to Yeast Mitochondrial and Phage T7 RNA Polymerases

Rui Sousa,Qing Guo,Dhananjaya Nayak

doi:10.1074/jbc.m900718200

Abstract

Yeast mitochondrial (YMt) and phage T7 RNA polymerases (RNAPs) are two divergent representatives of a large family of single subunit RNAPs that are also found in the mitochondria and chloroplasts of higher eukaryotes, mammalian nuclei, and many other bacteriophage. YMt and phage T7 promoters differ greatly in sequence and length, and the YMt RNAP uses an accessory factor for initiation, whereas T7 RNAP does not. We obtain evidence here that, despite these apparent differences, both the YMt and T7 RNAPs utilize a similar promoter recognition loop to bind their respective promoters. Mutations in this element in YMt RNAP specifically disrupt mitochondrial promoter utilization, and experiments with site-specifically tethered chemical nucleases indicate that this element binds the mitochondrial promoter almost identically to how the promoter recognition loop from the phage RNAP binds its promoter. Sequence comparisons reveal that the other members of the single subunit RNAP family display loops of variable sequence and size at a position corresponding to the YMt and T7 RNAP promoter recognition loops. We speculate that these elements may be involved in promoter recognition in most or all of these enzymes and that this element's structure allows it to accommodate significant sequence and length variation to provide a mechanism for rapid evolution of new promoter specificities in this RNAP family.

Highlights

The mechanisms of promoter recognition by the Yeast mitochondrial (YMt) and T7 RNA polymerases (RNAPs) appear to be distinct
It has been suggested that the YMt RNAP has a promoter recognition loop like that seen in T7 RNAP [12], and the recently described structure of N4 RNAP exhibits a promoter loop similar to that of T7 RNAP [13], this RNAP uses a hairpin promoter (Fig. 1) that is very different in structure from the T7 promoter
The sequences flanking this element are well conserved in this family and include the G, H, and I blocks of conserved elements previously identified in a comprehensive analysis of sequence conservation patterns in the single subunit RNAP family [11]

Summary

EXPERIMENTAL PROCEDURES

Enzyme and Template Preparations—Single cysteine-substituted mutants of YMt RNAP were constructed with the Stratagene PCR site-directed mutagenesis kit following manufacturer’s instructions. Transcription Assays—In vitro transcription reactions were carried out at room temperature for 15 min in 10 ␮l in transcription buffer (50 mM Tris-Cl (pH 8.0), 10 mM NaCl, 20 mM MgCl2, 10 mM dithiothreitol) containing a 0.5 mM concentration of each NTP (Trilink) and 0.1 ␮M duplex template or 0.01 ␮g/ml of poly(dI-dC). Transcription products were resolved by electrophoresis in 20% (w/v) polyacrylamide gels (19% acrylamide, 1% bisacrylamide, 7 M urea) in TBE buffer and analyzed with an Amersham Biosciences PhosphorImager. ICs halted at ϩ0, ϩ3, or ϩ7 were formed at room temperature in transcription buffer containing labeled bubble or duplex 14S rRNA template at 0.01 ␮M and Fe-BABE-conjugated YMt RNAP at 0.03 ␮M. Cleavage reactions were quenched after 5 s by the addition of one reaction volume of stop buffer (95% formamide, 20 mM EDTA, 0.1% xylene cyanol). Cleavage positions were mapped by reference to a Maxam-Gilbert G ϩ A ladder prepared as described [15]

RESULTS

DISCUSSION

Methods