Writing workshop Session-July 26 th 2019

1. Writing workshopSession-July 26th 2019 Linda S. Forero Quintero

2. Writing science. How to write papers that get cited and proposals that get funded. -> Ch. Condensing: • Tighten up your ideas and language. • Formatting tricks, but it can become a dense mass of text hard to understand. -> Strategies for condensing: “Prune the big limbs (1), then shake out the dead leaves(2)” (Roy Peter Clark) First figure out what you don’t need to say, and don’t say it. (Figure out your simple story) Second, cutting unnecessary words from the pieces that stay.

3. Targets for the delete key Obvious Redundancies

4. Targets for the delete key Modifiers: Adjectives and adverbs Metadiscourse: Talking about what you’re doing Verbosity

5. Let’s try condensing… The carboxyl-terminal domain (CTD) of RNA polymerase II (RNAP2) is dynamically phosphorylated during transcription in eukaryotic cells1-5. Even though visualization of transcriptional activity is possible at single6-8 and endogenous9-11 genes in living cells. The temporal ordering of phosphorylation only has been confirmed using tandem gene arrays12. However, because these studies are performed on a population of genes, the heterogeneity in dynamics along a single-copy gene remains unclear13. Here, we combined an optimized single-molecule microscope14 with fluorescent fragmented antibodies (Fabs)12 at an HIV-1 reporter gene8 to quantify the precise temporal order of phosphorylation at the CTD of endogenous RNAP2 at a single-copy gene. Our analyses of fluorescence fluctuations and run-off assay at the transcription site revealed that recruitment of unphosphorylated RNAP2 occurs first, followed by phosphorylation in the Serine 5 (Ser5ph), and subsequently in Serine 2 (Ser2ph) during active mRNA production. The strong correlations between single copy gene (MS2) and endogenous RNAP2 (Fabs) suggests the gene is not part of a larger transcription factory. Our global fit based on a unified mathematical framework predicted phosphorylation heterogeneity across a gene, arrival and departure times of RNAP2, time delays between transcription cycle steps, and our run-off data. We believe our methodology can now be extended to examine RNAP2 CTD phosphorylation dynamics at endogenous, unedited loci. The carboxyl-terminal domain (CTD) of RNA polymerase II (RNAP2) is dynamically phosphorylated during transcription in eukaryotic cells1-5. Although, visualization of transcriptional activity is possible at single6-8 and endogenous9-11 genes in living cells. The temporal ordering of phosphorylation only has been confirmed using tandem gene arrays12. However, because these studies are performed on a population of genes, the heterogeneity in dynamics along a single-copy gene remains unclear13. Here, we combined an optimized single-molecule microscope14 with fluorescent fragmented antibodies (Fabs)12 at an HIV-1 reporter gene8 to quantify the temporal order of phosphorylation at the CTD of endogenous RNAP2 at a single-copy gene. Fluorescence fluctuations and run-off assay revealed that recruitment of unphosphorylated RNAP2 occurs first, followed by phosphorylation in the Serine 5 (Ser5ph), and subsequently Serine 2 (Ser2ph) during mRNA synthesis. The strong correlations between HIV-1 and endogenous RNAP2 suggests the gene is not part of a larger transcription factory. Our global fit based on a unified mathematical framework predicted phosphorylation heterogeneity across a gene, RNAP2 phosphorylation temporality during the transcription cycle, and run-off data. We believe our methodology can now be extended to examine RNAP2 CTD phosphorylation dynamics at endogenous, unedited loci. 191 words 212 words!!! The aim is 180