Conservation of Promoter Melting Mechanisms in Divergent Regions of the Single-Subunit RNA Polymerases

Abstract

The single-subunit RNA polymerases are a widespread family of proteins found in phage, mitochondria, and chloroplasts. Unlike the phage RNAPs, the eukaryotic RNAPs require accessory factors to melt their promoters and diverge from the phage RNAPs in the regions where functions associated with promoter melting in the latter have been mapped, suggesting that promoter melting mechanisms in the eukaryotic RNAPs diverge from those in the phage enzymes. However, here we show that an element in the yeast mitochondrial RNAP, identified by sequence alignment with the T7 phage RNAP, fulfills a similar role in promoter melting as does the T7 RNAP ‘intercalating hairpin’. The yeast mitochondrial RNAP intercalating hairpin appears to be as important in promoter melting as is the mitochondrial transcription factor, MTF1, and both a structurally integral hairpin and MTF1 are required to achieve high levels of transcription on a duplex promoter. Deletions in the hairpin also relieve MTF1 inhibition of promoter escape on pre-melted promoters, likely because such deletions disrupt interactions with the upstream edge of the transcription bubble. These results are consistent with recent structural and functional studies of human mitochondrial RNAP and further reveal the surprising extent of mechanistic conservation between the eukaryotic and phage-encoded members of the single-subunit RNAP family.

The thumb subdomain of yeast mitochondrial RNA polymerase is involved in processivity, transcript fidelity and mitochondrial transcription factor binding

“…8, 9 Crystal structures of elongation complexes of T7RNAP show that residues R386 to R394 are involved in stabilizing a 7-base pair RNA:DNA hybrid, 22 whereas residues K767 to E788 Figure 5. ymtRNAP thumb mutants decrease synthesis of long transcripts.…”

“…9, lanes 2 and 3) or the thumb point mutants (data not shown) all the Mtf1 is present in the retenate. 9 The deletion mutants, however, were deficient in interacting with Mtf1 as all detectable Mtf1 was found in the filtrate (Fig. 8, lanes 6 and 9).…”

“…10 Wild-type and mutant ymtRNAPs were over-expressed in E. coli BL21 (DE3) Star and purified as previously described. 9 Transcription assays Transcription (run-off) reactions (15 mL volume) were conducted at room temperature for 15 min in 20 mM Tris-HCl (pH 8.0), 4 mM MgCl 2 , 10 mM DTT, and 50 mM NaCl (Transcription Buffer) with synthetic promoter templates at 0.05 mM, ymtRNAPs at 0.3 mM and Mtf1 (when present) at 1.8 mM, NTPs at 0.5 mM, and 0.1 mCi/mL of 3000 Ci/mM 32 P-a-ATP to label the transcripts. One to one volume of 98% formamide -100 mM EDTA (stop buffer) was used to stop the reaction.…”

“…As previously reported, wild-type ymtRNAP shifts the labeled probe at 1:1 molar ratio of Mtf1. 9 However, TH_D5 requires a 4-fold excess of Mtf1 to shift the probe, and TH_D11 requires a 16-fold excess. The apparent K d (measured as the interaction of the ymtRNAP-Mtf1 complex with a DNA template) for wild-type, TH_D5, and TH_D11 RNAPs on a duplex template were found to be 0.5 £ 10 ¡6 M, 1.5 £ 10 ¡6 M, and 5 £ 10 ¡6 M respectively (Fig.…”

“…4 Structural elements like the promoter recognition and DNA intercalating b-hairpin loops fulfill analogous functions in promoter recognition and opening in ymtRNAP and T7RNAP. [9][10][11] In contrast to phage RNAPs, mitochondrial RNAPs require accessory factors for promoter recognition, melting and termination. 12,13 T7 DNA and RNA polymerases achieve complex stability while allowing lateral movement via their thumb subdomains.…”