Biology subjects

Parilova, O.

Publications and source records attributed to Parilova, O..

2 recordsLinked to original sources

bioRxiv2024

Determinants of transcription initiation efficiency and start site selection by RNA polymerase I

RNA polymerase I (Pol I) synthesizes pre-ribosomal RNA, which is essential for ribosome biogenesis. Increased Pol I activity supports rapid cell growth, a key characteristic of cancer cells. Consequently, drugs targeting Pol I in cancer are being actively investigated. The first step in pre-rRNA synthesis involves the assembly of the Pol I transcription initiation complex on the rRNA gene promoter, yet the quantitative and dynamic parameters of this process remain inadequately defined. We combined biochemical, biophysical, and molecular dynamics simulation analyses to enhance molecular models illustrating how Pol I apparatus isolated Saccharomyces cerevisiae recognizes the promoter and the transcription start site (TSS). Time-resolved binding data shows that CF relies on a two-step mechanism, consisting of the binding and isomerization steps, to recognize its binding site on the promoter. The next step, CF-mediated recruitment of the Pol I- Rrn3 complex to the promoter, was inefficient, leading to the rapid dissociation of Pol I. The selection of the TSS by Pol I is influenced by the distance to CF and the properties of downstream DNA, such as bendability. The dynamics revealed in the Pol I transcription apparatus establish a framework for comparing the functions and mechanisms of Pol I activators and inhibitors.

biochemistry↗

bioRxiv2021

The formation of the bacterial RNA polymerase-promoter open complex involves a branched pathway

The expression of most bacterial genes commences with the binding of RNA polymerase (RNAP)-{sigma}70 holoenzyme to the promoter DNA. This initial RNAP-promoter closed complex undergoes a series of conformational changes, including the formation of a transcription bubble on the promoter and the loading of template DNA strand into the RNAP active site; these changes lead to the catalytically active open complex (RPO) state. Recent cryo-electron microscopy studies have provided detailed structural insight on the RPO and putative intermediates on its formation pathway. Here, we employ single-molecule fluorescence microscopy to interrogate the conformational dynamics and reaction kinetics during real-time RPO formation. We find that the RPO pathway is branched, generating RPO complexes with different stabilities. The RNAP cleft loops, and especially the {beta} rudder, stabilise the transcription bubble. The RNAP interactions with the promoter upstream sequence (beyond -35) stimulate transcription bubble nucleation and tune the reaction path towards stable forms of the RPO. The mechanistic heterogeneity of the RPO pathway may be a prerequisite for its regulation since such heterogeneity allows the amplification of small promoter sequence or transcription-factor-dependent changes in the free energy profile of the RPO pathway to large differences in transcription efficiency.

biochemistry↗