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.
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).
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.
Rosen, G.A., Baek, I., et al., Dynamics of RNA polymerase II and elongation factor Spt4/5 recruitment during activator-dependent transcription
DNA transcription is the most important nexus of gene regulation in all living organisms. In recent years, single-molecule experiments have given us a new window into the mechanisms of transcription and have revealed novel, previously unsuspected molecular behaviors and mechanisms. Ph.D. student Tim Harden used multi-color single-molecule fluorescence imaging to reveal a completely new transcription cycle for bacterial RNA polymerase. Harden and co-authors showed that an RNA polymerase molecule in vitro frequently (in >90% of transcription events) remains bound to DNA and may again initiate transcription after it has terminated the first round of transcription. Even more unexpectedly, this “secondary initiation” is not restricted to the same RNA. After the first round, the polymerase can scan thousands of basepairs along the DNA and can initiate at a different start site, frequently one that is oriented in the opposite direction and produces an antisense transcript.
To complement the single-molecule studies in vitro, the manuscript reports new analyses of whole-transcriptome cellular RNAs revealed by the Rend-seq method that measures transcript initiation and termination frequencies across the genome with single basepair resolution. These provide evidence that the new transcription cycle may be responsible for initiating antisense transcription at hundreds of genomic locations in the two widely divergent bacterial species examined. The work defines a new mechanism for the regulated production of antisense RNAs, many of which are now recognized as important agents of gene-specific regulation through control of transcription, mRNA decay, and translation. In addition, the new transcription cycle provides a mechanism through which transcription initiation can be controlled not just through feedback networks involving multiple genes, but also through production of multiple different primary transcripts consequent to a single RNA polymerase-to-DNA recruitment event.
Harden, T.T., et al. Alternative transcription cycle for bacterial RNA polymerase.
Nature Communications 11, 450 (2020).
RNA polymerases contain a conserved “secondary channel” in which proteins that regulate transcription can bind. In Escherichia coli bacteria, the secondary channel factors (SCFs) GreB and DksA both repress initiation of ribosomal RNA synthesis, but SCF loading and repression mechanisms are unclear. Sarah Stumper and her collaborators used fluorescence correlation spectroscopy and multi-wavelength single-molecule fluorescence colocalization microscopy to show that the SCFs likely repress transcription through an interesting “delayed inhibition” mechanism in which the proteins arrive at DNA already complexed to RNA polymerase and block at a later stage of transcription initiation. The work explains factors that control the relative contributions of the two proteins to regulation and suggests a mechanism by which repression is restricted to ribosomal RNA and other promoters that form short-duration complexes with RNA polymerase.
Delayed inhibition mechanism for secondary channel factor regulation of ribosomal RNA transcription.
Stumper, S.K., Ravi, H., Friedman, L.J., Mooney, R.A., Corrêa, I.R., Gershenson, A., Landick, R., and Gelles, J.
eLife (2019) 8:e40576
In living cells, messenger RNAs are not manufactured by RNA polymerases (RNAPs) functioning alone. Instead, RNA synthesis is carried out collectively by RNAP together with accessory proteins that associate with the RNAP-containing transcription elongation complex and modulate its activity. In this paper, Larry Tetone, Larry Friedman, and Melissa Osborne, along with their collaborators from the Gelles and Landick labs, used multi-wavelength single-molecule fluorescence methods to for the first time directly observe the dynamic binding and dissociation of an accessory protein with an RNAP during active transcript elongation. The protein, GreB, is important for transcript proofreading in E. coli and other bacteria and is a functional analog of the TFIIS protein in eaukaryotes. “Unexpectedly,” the authors report, “GreB was not selectively recruited to RNAPs requiring its transcript proofreading function. Instead, GreB transiently bound to multiple types of complexes, eventually finding via random search RNAPs that require its activity. The observations suggest a paradigm by which a regulator can act while minimizing obstruction of a binding site that must be shared with other proteins.”
Dynamics of GreB-RNA polymerase interaction allow a proofreading accessory protein to patrol for transcription complexes needing rescue
Larry E. Tetone, Larry J. Friedman, Melisa L. Osborne, Harini Ravi, Scotty Kyzer, Sarah K. Stumper, Rachel A. Mooney, Robert Landick, and Jeff Gelles
PNAS (2017) 114:E1081-E1090.
New plasmids reported in this article can be obtained from Addgene