Dataset Information

Identifying a core RNA polymerase surface critical for interactions with a sigma-like specificity factor.

ABSTRACT: Cyclic interactions occurring between a core RNA polymerase (RNAP) and its initiation factors are critical for transcription initiation, but little is known about subunit interaction. In this work we have identified regions of the single-subunit yeast mitochondrial RNAP (Rpo41p) important for interaction with its sigma-like specificity factor (Mtf1p). Previously we found that the whole folded structure of both polypeptides as well as specific amino acids in at least three regions of Mtf1p are required for interaction. In this work we started with an interaction-defective point mutant in Mtf1p (V135A) and used a two-hybrid selection to isolate suppressing mutations in the core polymerase. We identified suppressors in three separate regions of the RNAP which, when modeled on the structure of the closely related phage T7 RNAP, appear to lie on one surface of the protein. Additional point mutations and biochemical assays were used to confirm the importance of each region for Rpo41p-Mtf1p interactions. Remarkably, two of the three suppressors are found in regions required by T7 RNAP for DNA sequence recognition and promoter melting. Although these essential regions of the phage RNAP are poorly conserved with the mitochondrial RNAPs, they are conserved among the mitochondrial enzymes. The organellar RNAPs appear to use this surface in an alternative way for interactions with their separate sigma-like specificity factor, which, like its bacterial counterpart, provides promoter recognition and DNA melting functions to the holoenzyme.

SUBMITTER: Cliften PF

PROVIDER: S-EPMC88776 | biostudies-literature | 2000 Sep

REPOSITORIES: biostudies-literature

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Identifying a core RNA polymerase surface critical for interactions with a sigma-like specificity factor.

Cliften P F PF Jang S H SH Jaehning J A JA

Molecular and cellular biology 20000901 18

Cyclic interactions occurring between a core RNA polymerase (RNAP) and its initiation factors are critical for transcription initiation, but little is known about subunit interaction. In this work we have identified regions of the single-subunit yeast mitochondrial RNAP (Rpo41p) important for interaction with its sigma-like specificity factor (Mtf1p). Previously we found that the whole folded structure of both polypeptides as well as specific amino acids in at least three regions of Mtf1p are re ...[more]

PMID: 10958696

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Project description:The canonical function of a bacterial sigma (σ) factor is to determine the gene specificity of the RNA polymerase (RNAP). In several diverse bacterial species, the σ54 factor uniquely confers distinct functional and regulatory properties on the RNAP. A hallmark feature of the σ54-RNAP is the obligatory requirement for an activator ATPase to allow transcription initiation. Different activator ATPases couple diverse environmental cues to the σ54-RNAP to mediate adaptive changes in gene expression. Hence, the genes that rely upon σ54 for their transcription have a wide range of different functions suggesting that the repertoire of functions performed by genes, directly or indirectly affected by σ54, is not yet exhaustive. By comparing the growth patterns of prototypical enteropathogenic, uropathogenic, and nonpathogenic Escherichia coli strains devoid of σ54, we uncovered that the absence of σ54 results in two differently sized colonies that appear at different times specifically in the uropathogenic E. coli (UPEC) strain. Notably, UPEC bacteria devoid of individual activator ATPases of the σ54-RNAP do not phenocopy the σ54 mutant strain. Thus, it seems that σ54's role as a determinant of uniform colony appearance in UPEC bacteria represents a putative non-canonical function of σ54 in regulating genetic information flow. IMPORTANCE RNA synthesis is the first step of gene expression. The multisubunit RNA polymerase (RNAP) is the central enzyme responsible for RNA synthesis in bacteria. The dissociable sigma (σ) factor subunit directs the RNAP to different sets of genes to allow their expression in response to various cellular needs. Of the seven σ factors in Escherichia coli and related bacteria, σ54 exists in a class of its own. This study has uncovered that σ54 is a determinant of the uniform growth of uropathogenic E. coli on solid media. This finding suggests a role for this σ54 in gene regulation that extends beyond its known function as an RNAP gene specificity factor.

Dataset Information

Identifying a core RNA polymerase surface critical for interactions with a sigma-like specificity factor.

Publications

Identifying a core RNA polymerase surface critical for interactions with a sigma-like specificity factor.

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