Tag Archives: NCI-funded

Single-molecule studies reveal branched pathways for activator-dependent assembly of RNA polymerase II pre-initiation complexes

A key event leading to the synthesis of a eukaryotic messenger RNA  is the assembly of a pre-initiation complex (PIC) on promoter DNA near the transcription start site.  The PIC contains RNA polymerase II (pol II) plus general transcription factors TFIID, TFIIA, TFIIB, TFIIF, TFIIE, and TFIIH.  PIC assembly is enhanced by binding of transcription activator proteins to separate DNA sites called upstream activating sequences (UAS) or enhancers, but the dynamic mechanisms by which activators at other sites control PIC assembly has been unclear.  In this paper, Inwha Baek (from the Buratowski lab) and Larry Friedman (from the Gelles lab) defined this mechanism using multi-wavelength single-molecule fluorescence microscopy.  The experiments used budding yeast nuclear extract with fluorescently labeled proteins and the strong artificial activator protein Gal4-VP16.  The investigators found that, unexpectedly, pol II and often TFIIE and TFIIF were not recruited directly to the promoter.  Instead they first bound via the activator to the UAS and were then subsequently transferred, likely as a pre-formed complex, to the promoter.  This work gives new insight into how messenger RNA synthesis is regulated to switch genes on and off in eukaryotic cells.  It also suggests how multiple pol II molecules may be poised at UAS sequences ready to transcribe an adjacent gene, which may explain some of the “bursts” of transcription detected in living cells.

10.1016/j.molcel.2021.07.025
Baek I., et al., Single-molecule studies reveal branched pathways for activator-dependent assembly of RNA polymerase II pre-initiation complexes
Molecular Cell 81, 3576-3588.e6 (2021).

Dynamics of RNA polymerase II and elongation factor Spt4/5 recruitment during activator-dependent transcription

DNA transcription by RNA polymerase II (RNApII) is arguably the process most central to regulation of gene expression in eukaryotic organisms.  Regulated transcription requires the formation on DNA of molecular assemblies containing not only RNApII but also dozens of accessory proteins that play pivotal roles in the process.  While we know about the structures of some of these assemblies in atomic detail, quantitative understanding of the dynamics and pathways by which the assemblies interconvert and progress through this fundamental gene expression pathway is largely lacking.

In this study we report single-molecule fluorescence microscopy studies of transcription in yeast nuclear extract, for the first time visualizing and measuring the dynamics of activator-dependent recruitment of RNApII and the central elongation factor Spt4/5 to transcription complexes.  Grace Rosen (Jeff Gelles’ labortatory, Brandeis) , Inwha Baek (Steve, Buratowski’s lab, Harvard Medical School), and collaborators elucidated the kinetically significant steps in activated RNApII transcription initiation and show for the first time that Spt4/5 dynamics are tuned to the typical lifetimes of transcription elongation complexes.  In addition to these substantive results, our work represents an important methodological advance.  As the first application of the CoSMoS (co-localization single-molecule spectroscopy) technique to activated eukaryotic transcription, it demonstrates a general method for elucidating the correlated dynamic interactions of different components of the machinery with initiation and elongation transcription complexes.  The approach is likely to find further use in studies of the mechanistic features of RNApII transcription.

https://doi.org/10.1073/pnas.2011224117
Rosen, G.A., Baek, I., et al., Dynamics of RNA polymerase II and elongation factor Spt4/5 recruitment during activator-dependent transcription
PNAS