Live imaging of transcription sites using an elongating RNA polymerase II–specific probe

In eukaryotic nuclei, most genes are transcribed by RNA polymerase II (RNAP2), whose regulation is a key to understanding the genome and cell function. RNAP2 has a long heptapeptide repeat (Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7), and Ser2 is phosphorylated on an elongation form. To detect RNAP2 Ser2 phosphorylation (RNAP2 Ser2ph) in living cells, we developed a genetically encoded modification-specific intracellular antibody (mintbody) probe. The RNAP2 Ser2ph-mintbody exhibited numerous foci, possibly representing transcription “factories,” and foci were diminished during mitosis and in a Ser2 kinase inhibitor. An in vitro binding assay using phosphopeptides confirmed the mintbody’s specificity. RNAP2 Ser2ph-mintbody foci were colocalized with proteins associated with elongating RNAP2 compared with factors involved in the initiation. These results support the view that mintbody localization represents the sites of RNAP2 Ser2ph in living cells. RNAP2 Ser2ph-mintbody foci showed constrained diffusional motion like chromatin, but they were more mobile than DNA replication domains and p300-enriched foci, suggesting that the elongating RNAP2 complexes are separated from more confined chromatin domains.

Visualizing transcription sites in living cells using a genetically encoded probe specific for the elongating form of RNA polymerase II

In eukaryotic nuclei, most genes are transcribed by RNA polymerase II (RNAP2). How RNAP2 transcription is regulated in the nucleus is a key to understanding the genome and cell function. The largest subunit of RNAP2 has a long heptapeptide repeat (Tyr1-Ser2-Pro3-Thr4-Ser5- Pro6-Ser7) at the C-terminal domain and Ser2 is phosphorylated on an elongation form of RNAP2. To detect RNAP2 Ser2 phosphorylation (RNAP2 Ser2ph) in living cells, we developed a genetically encoded modification-specific intracellular antibody (mintbody) probe. The RNAP2 Ser2ph-mintbody probe exhibited numerous foci, possibly representing transcription “factories” in living HeLa cells, and foci were diminished when cells were treated with triptolide to induce RNAP2 degradation and with flavopiridol to inhibit Ser2ph. An in vitro binding assay using phospho-peptides confirmed the Ser2ph-specific binding of the mintbody. These results support the view that mintbody localization represents the sites of RNAP2 Ser2ph in living cells. RNAP2 Ser2ph-mintbody foci were colocalized with proteins associated with elongating RNAP2, such as the CDK12 and Paf1 complex component, compared to factors involved in transcription activation around the transcription start sites, such as CDK9 and BRD4. Tracking analysis revealed that RNAP2 Ser2ph-mintbody foci showed constrained diffusional motion like chromatin, but was more mobile compared to euchromatin domains, suggesting that the elongating RNAP2 complexes are separated from the more confined initiating clusters.SummaryThe authors developed a genetically encoded probe to specifically detect the Ser2- phosphorylated, elongating form of RNA Polymerase II in living cells. The motion of Ser2- phosphorylated polymerase foci was more dynamic than chromatin domains, suggesting that the elongating complexes are separated from the more confined initiating clusters.

Reagentless Biomolecular Analysis Using a Nanoscale Molecular Pendulum

The ability to sense biological inputs using self-contained devices unreliant on external reagents or reporters would open countless opportunities to collect information about our health and environment. Currently, a very limited set of molecular inputs can be detected using this type of sensor format. The development of versatile reagentless sensors that could track molecular analytes in biological fluids remains an unmet need. Here, we describe a new universal sensing mechanism that is compatible with the analysis of proteins that are important physiological markers of stress, allergy, cardiovascular health, inflammation and cancer. The sensing mechanism we developed is based on the measurement of field-induced directional diffusion of a nanoscale molecular pendulum tethered to an electrode surface and the sensitivity of electron-transfer reaction kinetics to molecular size. Using time-resolved electrochemical measurements of diffusional motion, the presence of an analyte bound to a sensor complex can be continuously tracked in real time. We show that this sensing approach is compatible with making measurements in blood, saliva, urine, tears and sweat and that the sensors can collect data in situ in living animals. The sensor platform described enables a broad range of applications in personalized health monitoring.

Quantifying active diffusion in an agitated fluid

Single-particle tracking reveals an enhanced diffusional motion of tracer beads when agitating the surrounding fluid with miniaturized magnetic stir bars. Signatures of the stirring are mostly encoded in correlation functions of the particle motion.