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Conservation of promoter melting mechanisms in divergent regions of the single-subunit RNA polymerases.

ABSTRACT: The single-subunit RNA polymerases make up 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 role in promoter melting similar to that filled by the T7RNAP "intercalating hairpin". The yeast mitochondrial RNAP intercalating hairpin appears to be as important in promoter melting as 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 from the hairpin also relieve MTF1 inhibition of promoter escape on premelted 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.

SUBMITTER: Velazquez G

PROVIDER: S-EPMC3361065 | biostudies-literature | 2012 May

REPOSITORIES: biostudies-literature

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Conservation of promoter melting mechanisms in divergent regions of the single-subunit RNA polymerases.

Velazquez Gilberto G Guo Qing Q Wang Liping L Brieba Luis G LG Sousa Rui R

Biochemistry 20120427 18

The single-subunit RNA polymerases make up 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 ...[more]

PMID: 22524540

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Project description:Key messageWe identified sequence motifs, which enhance or reduce the ability of the Arabidopsis phage-type RNA polymerases RPOTm (mitochondrial RNAP), RPOTp (plastidial RNAP), and RPOTmp (active in both organelles) to recognize their promoters in vitro with help of a 'specificity loop'. The importance of this data for the evolution and function of the organellar RNA polymerases is discussed. The single-subunit RNA polymerase (RNAP) of bacteriophage T7 is able to perform all steps of transcription without additional transcription factors. Dicotyledonous plants possess three phage-type RNAPs, RPOTm-the mitochondrial RNAP, RPOTp-the plastidial RNAP, and RPOTmp-an RNAP active in both organelles. RPOTm and RPOTp, like the T7 polymerase, are able to recognize promoters, while RPOTmp displays no significant promoter specificity in vitro. To find out which promoter motifs are crucial for recognition by the polymerases we performed in vitro transcription assays with recombinant Arabidopsis RPOTm and RPOTp enzymes. By comparing different truncated and mutagenized promoter constructs, we observed the same minimal promoter sequence supposed to be needed in vivo for transcription initiation. Moreover, we identified elements of core and flanking sequences, which are of critical importance for promoter recognition and activity in vitro. We further intended to reveal why RPOTmp does not efficiently recognize promoters in vitro and if promoter recognition is based on a structurally defined specificity loop of the plant enzymes as described for the yeast and T7 RNAPs. Interestingly, the exchange of only three amino acids within the putative specificity loop of RPOTmp enabled the enzyme for specific promoter transcription in vitro. Thus, also in plant phage-type RNAPs the specificity loop is engaged in promoter recognition. The results are discussed with respect to their relevance for transcription in organello and to the evolution of RPOT enzymes including the divergence of their functions.

Dataset Information

Conservation of promoter melting mechanisms in divergent regions of the single-subunit RNA polymerases.

Publications

Conservation of promoter melting mechanisms in divergent regions of the single-subunit RNA polymerases.

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