Picocalorimetry of Transcription by RNA Polymerase

Thermal variations can exert dramatic effects on the rates of enzymes. The influence of temperature on RNA polymerase is of particular interest because its transcriptional activity governs general levels of gene expression, and may therefore exhibit pleiotropic effects in cells. Using a custom-modified optical trapping apparatus, we used a tightly focused infrared laser to heat single molecules of Escherichia coli RNA polymerase while monitoring transcriptional activity. We found a significant change in rates of transcript elongation with temperature, consistent with a large enthalpic barrier to the condensation reaction associated with RNA polymerization (∼13 kcal/mol). In contrast, we found little change in either the frequency or the lifetime of off-pathway, paused states, indicating that the energetic barrier to transcriptional pausing is predominantly entropic

Direct observation of base-pair stepping by RNA polymerase.

During transcription, RNA polymerase (RNAP) moves processively along a DNA template, creating a complementary RNA. Here we present the development of an ultra-stable optical trapping system with ångström-level resolution, which we used to monitor transcriptional elongation by single molecules of Escherichia coli RNAP. Records showed discrete steps averaging 3.7^0.6 Å , a distance equivalent to the mean rise per base found in B-DNA. By combining our results with quantitative gel analysis, we conclude that RNAP advances along DNA by a single base pair per nucleotide addition to the nascent RNA. We also determined the force-velocity relationship for transcription at both saturating and subsaturating nucleotide concentrations; fits to these data returned a characteristic distance parameter equivalent to one base pair. Global fits were inconsistent with a model for movement incorporating a power stroke tightly coupled to pyrophosphate release, but consistent with a brownian ratchet model incorporating a secondary NTP binding site

Global DNA Compaction in Stationary-Phase Bacteria Does Not Affect Transcription

In stationary-phase Escherichia coli, Dps (DNA-binding protein from starved cells) is the most abundant protein component of the nucleoid. Dps compacts DNA into a dense complex and protects it from damage. Dps has also been proposed to act as a global regulator of transcription. Here, we directly examine the impact of Dps-induced compaction of DNA on the activity of RNA polymerase (RNAP). Strikingly, deleting the dps gene decompacted the nucleoid but did not significantly alter the transcriptome and only mildly altered the proteome during stationary phase. Complementary in vitro assays demonstrated that Dps blocks restriction endonucleases but not RNAP from binding DNA. Single-molecule assays demonstrated that Dps dynamically condenses DNA around elongating RNAP without impeding its progress. We conclude that Dps forms a dynamic structure that excludes some DNA-binding proteins yet allows RNAP free access to the buried genes, a behavior characteristic of phase-separated organelles. Despite markedly condensing the bacterial chromosome, the nucleoid-structuring protein Dps selectively allows access by RNA polymerase and transcription factors at normal rates while excluding other factors such as restriction endonucleases.</p