DNA-binding transcription factors (TFs) remain challenging to target with molecular probes. Many TFs function in part through interaction with Mediator, a 26-subunit complex that controls RNA polymerase II activity genome-wide. We sought to block p53 function by disrupting the p53-Mediator interaction. Through rational design and activity-based screening, we characterize a stapled peptide, with functional mimics of both p53 activation domains, that blocks p53-Mediator binding and selectively inhibits p53-dependent transcription in human cells; importantly, this "bivalent" peptide has negligible impact, genome-wide, on non-p53 target genes. Our proof-of-concept strategy circumvents the TF entirely and targets the TF-Mediator interface instead, with desired functional outcomes (i.e., selective inhibition of p53 activation). Furthermore, these results demonstrate that TF activation domains represent viable starting points for Mediator-targeting molecular probes, as an alternative to large compound libraries. Different TFs bind Mediator through different subunits, suggesting this strategy could be broadly applied to selectively alter gene expression programs.

CDK7 phosphorylates the RNA polymerase II (pol II) C-terminal domain CTD and activates the P-TEFb-associated kinase CDK9, but its regulatory roles remain obscure. Here, using human CDK7 analog-sensitive (CDK7as) cells, we observed reduced capping enzyme recruitment, increased pol II promoter-proximal pausing, and defective termination at gene 3' ends upon CDK7 inhibition. We also noted that CDK7 regulates chromatin modifications downstream of transcription start sites. H3K4me3 spreading was restricted at gene 5' ends and H3K36me3 was displaced toward gene 3' ends in CDK7as cells. Mass spectrometry identified factors that bound TFIIH-phosphorylated versus P-TEFb-phosphorylated CTD (versus unmodified); capping enzymes and H3K4 methyltransferase complexes, SETD1A/B, selectively bound phosphorylated CTD, and the H3K36 methyltransferase SETD2 specifically bound P-TEFb-phosphorylated CTD. Moreover, TFIIH-phosphorylatedCTDstimulated SETD1A/B activity toward nucleosomes, revealing a mechanistic basis for CDK7 regulation of H3K4me3 spreading. Collectively, these results implicate a CDK7-dependent "CTD code'' that regulates chromatin marks in addition to RNA processing and pol II pausing.

Initiation is a highly regulated, rate-limiting step in transcription. We employed a series of approaches to examine the kinetics of RNA polymerase (RNAP) transcription initiation in greater detail. Quenched kinetics assays, in combination with magnetic tweezer experiments and other methods, showed that, contrary to expectations, RNAP exit kinetics from later stages of initiation (e.g. from a 7-base transcript) was markedly slower than from earlier stages. Further examination implicated a previously unidentified intermediate in which RNAP adopted a long-lived backtracked state during initiation. In agreement, the RNAP-GreA endonuclease accelerated transcription kinetics from otherwise delayed initiation states and prevented RNAP backtracking. Our results indicate a previously uncharacterized RNAP initiation state that could be exploited for therapeutic purposes and may reflect a conserved intermediate among paused, initiating eukaryotic enzymes. Significance: Transcription initiation by RNAP is rate limiting owing to many factors, including a newly discovered slow initiation pathway characterized by RNA backtracking and pausing. This backtracked and paused state occurs when all NTPs are present in equal amounts, but becomes more prevalent with NTP shortage, which mimics cellular stress conditions. Pausing and backtracking in initiation may play an important role in transcriptional regulation, and similar backtracked states may contribute to pausing among eukaryotic RNA polymerase II enzymes.

Initiation is a highly regulated, rate-limiting step in transcription. We used a series of approaches to examine the kinetics of RNA polymerase (RNAP) transcription initiation in greater detail. Quenched kinetics assays, in combination with gel-based assays, showed that RNAP exit kinetics from complexes stalled at later stages of initiation (e.g., from a 7-base transcript) were markedly slower than from earlier stages (e.g., from a 2- or 4-base transcript). In addition, the RNAP-GreA endonuclease accelerated transcription kinetics from otherwise delayed initiation states. Further examination with magnetic tweezers transcription experiments showed that RNAP adopted a long-lived backtracked state during initiation and that the paused-backtracked initiation intermediate was populated abundantly at physiologically relevant nucleoside triphosphate (NTP) concentrations. The paused intermediate population was further increased when the NTP concentration was decreased and/or when an imbalance in NTP concentration was introduced (situations that mimic stress). Our results confirm the existence of a previously hypothesized paused and backtracked RNAP initiation intermediate and suggest it is biologically relevant; furthermore, such intermediates could be exploited for therapeutic purposes and may reflect a conserved state among paused, initiating eukaryotic RNA polymerase II enzymes.

Promoter-proximal RNA Polymerase II (Pol-II) pausing has been shown to play a significant role in transcription regulation of elongating Pol-II complexes in a large number of metazoan and mammalian genes (1). The traditional understanding of transcription regulation in mammals involved controlling Pol-II recruitment to promoters and controlling initial steps at the promoter, including pre-initiation complex formation and promoter escape. Most works investigating promoter-proximal PolII pausing have employed chromatin immunoprecipitation followed by sequencing to determine Pol-II localization or in vitro transcriptional assays using nuclear extracts analyzed with radioactive gel electrophoresis. In order to gain greater mechanistic insight into the regulation of promoter-proximal Pol-II pausing, we use single molecule ALEX spectroscopy to monitor RNA transcripts production as function of composition and order of addition of transcription factors to an in vitro reconstituted human Pol-II system. The RNA transcripts are detected by complementary doubly dye-labeled single-stranded DNA (ssDNA) probes. The human gene HSPA1B for heat shock protein 70 (Hsp70) is used as a model system due to its extensive characterization in drosophila. Our approach provides a rapid, sensitive and robust avenue for screening protein factors regulating promoter-proximal Pol-II pausing. 1. H. Kwak, J. T. Lis, Control of transcriptional elongation. Annu. Rev. Genet. 47, 483-508 (2013).

An essential and highly regulated step in gene expression is transcription initiation. After promoter binding and DNA unwinding ('bubble opening') and in the presence of nucleoside triphosphates (NTPs), the RNA polymerase (RNAP)-promoter initial transcribing complex (RPitc) engages in 'abortive initiation', a process in which RNAP cycles between synthesis and release of short RNA transcripts. In abortive initiation, RPitc is believed to undergo a sequence of transitions between different initiation sub-states. The kinetics of the production of a full RNA transcript starting at a late initiation sub-state is expected to be similar or faster than the kinetics measured from an earlier initiation sub-state. To test this hypothesis, we developed a novel in vitro single-run quenched kinetics transcription assay based on the detection and quantification of run-off transcripts. Using this assay and corroborating it with gel-based and magnetic tweezer assays, testing two different promoters, we surprisingly found that run-off transcription kinetics starting from late initiation sub-states is slower than kinetics starting from earlier initiation sub-states. When the same kinetic measurements were performed in the presence of the transcription elongation factor GreA, the kinetics starting from a late initiation sub-state was accelerated. Experimental results suggest that as a function of shortage in NTPs RPitc can enter an off-pathway state in which the nascent RNA is in a backtracked, paused position, awaiting incorporation of the missing NTP. Our findings suggest that pausing at distinct stages of transcription initiation could regulate gene expression under stressed conditions.

Key Points A basic function of the Mediator complex is to communicate regulatory signals from DNA-binding transcription factors (TFs) directly to RNA polymerase II (Pol II). Different TFs, and the signalling pathways that regulate these TFs, often interact with different Mediator subunits to regulate expression of their target genes. Mediator is composed of a large number of subunits, some of which can reversibly associate with Mediator or are expressed at variable levels in different cell types. Mediator binding to various proteins and protein complexes, such as TFs, Pol II and the cyclin-dependent kinase 8 (CDK8) module, results in large-scale structural changes. These structural changes, in turn, appear to modulate the function of Mediator and may affect its ability to bind to other proteins. Because of its direct and extensive interactions with Pol II, Mediator regulates multiple stages of Pol II transcription (for example, initiation and re-initiation). Mediator interactions with the super elongation complex (SEC) also seem to be important for its regulation of Pol II elongation. The interactions between TFs, Mediator, cohesin and the pre-initiation complex (PIC) correlate with the formation of enhancer–promoter DNA loops, which are an important regulatory mechanism. Interactions between Mediator and non-coding RNA also correlate with DNA looping.