Abstract
Promoter recognition is the first and the most important step during gene expression. Our studies of the yeast (Saccharomyces cerevisiae) mitochondrial (mt) transcription machinery provide mechanistic understandings on the basic problem of how the mt RNA polymerase (RNAP) with the help of the initiation factor discriminates between promoter and non-promoter sequences. We have used fluorescence-based approaches to quantify DNA binding, bending, and opening steps by the core mtRNAP subunit (Rpo41) and the transcription factor (Mtf1). Our results show that promoter recognition is not achieved by tight and selective binding to the promoter sequence. Instead, promoter recognition is achieved by an induced-fit mechanism of transcription factor-dependent differential conformational changes in the promoter and non-promoter DNAs. While Rpo41 induces a slight bend upon binding both the DNAs, addition of the Mtf1 results in severe bending of the promoter and unbending of the non-promoter DNA. Only the sharply bent DNA results in the catalytically active open complex. Such an induced-fit mechanism serves three purposes: 1) assures catalysis at promoter sites, 2) prevents RNA synthesis at non-promoter sites, and 3) provides a conformational state at the non-promoter sites that would aid in facile translocation to scan for specific sites.
Highlights
We have investigated the transcription machinery of the yeast (Saccharomyces cerevisiae) mitochondria, which consists of a nuclear encoded ϳ153 kDa core subunit Rpo41 [12] and a ϳ45 kDa transcription factor Mtf1 [13, 14]
Rpo41-Mtf1 induces selective DNA bending in the promoter and unbending in the non-promoter DNA, based on which we propose that promoter selection is achieved by an induced-fit mechanism involving differential conformational changes
We have investigated how the transcription machinery of the yeast mitochondria consisting of the catalytic subunit Rpo41 and the transcription factor Mtf1 selectively initiates transcription from the promoter sequence and discriminate against the non-promoter sequences
Summary
We have investigated the transcription machinery of the yeast (Saccharomyces cerevisiae) mitochondria, which consists of a nuclear encoded ϳ153 kDa core subunit Rpo41 [12] and a ϳ45 kDa transcription factor Mtf1 [13, 14]. To determine the role of the core subunit and the transcription factor and the mechanism of promoter selection, we have quantified the interactions of Rpo41 and Rpo41-Mtf1 with the promoter and non-promoter DNAs and characterized the protein-induced structural changes in these DNAs. Using a combination of fluorescence-based methods including fluorescence anisotropy, Forster resonance energy transfer (FRET), and 2-aminopurine (2-AP) fluorescence, we have measured DNA binding, bending, and melting.
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.