Compartmentalization by directional gene expression

1. Student ID: 250599864 Compartmentalization by directional gene expression Shirley S. Daube, Dan Bracha, AmnonBuxboim, and Roy H. Bar-Zivb Department of Materials and Interfaces, The Weizmann Institute of Science, Rehovot, Israel, 76100 • Introduction • Compartmentalization plays a role in establishing macromolecular and solvent gradients • Previous research shows that bacterial genome is separated from cytoplasm (Zimmerman 2006. J StructBiol 156: 255) and transcription in the nucleus of eukaryotes occurs in non-random order, with position of the genes influencing their expression (Gondor, Ohlsson2009. Nature 461: 212; Sutherland, Bickmore 2009. Nat Rev Genet 10: 457) • The highly compartmentalized and crowded character that is typical for the environment of DNA found in living cells is hard to reproduce in vitro • Objective • Explore how gene expression is affected by density, spatial heterogeneity and arrangement of the DNA and the environment (comparing between transcription reaction in dilute DNA solution and in DNA brush) • Try to establish the parameters leading to barrier-free compartmenta- lizationof transcription reaction from the reservoir in the DNA brush • Results • TX reaction in DNA brushes (with TX unit in a Mid/IN or Mid/OUT position) vs. in dilute DNA solution • RNA synthesis increased linearly with time in both cases • In DNA brush the reaction slowed down after 1hour (giving ∼ 50 RNAs per each DNA template) (Fig. 2A) • Increasing the total RNAP concentration resulted in linear increase in RNA production with brush rate reaching saturation at ∼10 μM RNAP (no consequent rNTP depletion) • In dilute solution the rate did not reach saturation and was ∼10 times higher that in the brush (limited only by depletion of rNTPs) • Density of DNA brush inhibits TX • Increasing brush density caused increase in volume exclusion thereby lowering the number of rNTPs present • DenseMid/IN brushes with high number of coding DNA (contained promoter) were constructed (Fig. 3A) • TX rate per promoter was almost constant at low densities and decreased at high density (Fig. 3B) • Mixed DNA brushes with additional non-coding DNA added achieved constant TX efficiency (Fig. 3B) • The TX rate for coding DNA brushes was higher than for mixed DNA brushes • Results imply that non-coding DNA reduces TX rate due to increasing brush density and consequent exclusion of rNTPs • Orientation of TX within a brush • TX rate in dilute DNA solution was constant in all 6 promoter arrangements (Fig. 1B). • The TX rates were markedly different for all confiigurations of the dense DNA brush (coding DNA only) (Fig. 4A) • TX rate was always higher for IN than OUT arrangements (at all distances) • Addition of spermidine (polyamine, used to stimulate RNAP activity) accentuated the IN/OUT ratio (Fig. 4B) • Local rNTP gradient was formed (driven by rNTP hydrolysis) producing a sink (promoter) and a source (terminator) for RNAP • For IN configuration the active gradient worked against the passive gradient – which increased the RNAP local concentration • For Out Brush the gradient contributed to repulsive forces and the RNAP concentration decreased • Methods • Synthetic DNA brush • Linear, end-bound DNA polymers lined up along horizontal layers with set mean distance (30 nm) and alignment relative to the surface (Fig. 1A) • 2.1-kbp-long DNA fragments with a promoter, terminator and a 280-bp-long flanking DNA segment (transcription unit) • 6 arrangements evaluated – at 3 distances from the surface (Bottom, Mid, Top), pointing towards (IN) or from the surface (OUT) (Fig. 1B) • Transcription (TX) • Carried out by T7 RNA polymerase (RNAP) • 10 μL reaction mix (RNAP, NTPs, and buffer) was added to a DNA brush chamber (3-mm-diameter), thereby initiating the TX reaction. • Same reaction mix was used for solution with free DNA • Reaction was stopped by removing 6 μL from the chamber and adding them to 1.5 μL of a 5× stop solution to obtain final solution of 0.2% SDS/20 mMEDTA, and cooled on ice Fig. 2. (A) TX rate in DNA brush as function over time (at 2 μM RNAP), showing a decline in rate after 60 min. Inset: TX rate over time in dilute solution (equal conditions). (B) TX rate in DNA brush as function of RNAP concentration. Inset: TX vs. [RNAP] in dilute solution (equal conditions). Fig. 4. (A) Effect of promoter orientation and position on TX rate in 6 brush arrangements (without spermidine). (B) Relationship between the IN/OUT ratio of rates (with/without spermidine) and the promoter distance from the surface (in bps) (OUT configuration only). • Conclusions • Experimental data provided the information needed to answer the research questions: • Using a surface-bound DNA for TX reaction creates a segregated environment that differs in nature from the surrounding solution • Results indicate that TX reaction in the DNA brush is dependant on density of the brush (high density decreases TX rate) as well as the position and direction of the promoter (favoring IN direction) • Based on the findings, it can be proposed that RNAP concentration in the brush is related to: • - brush density (high density excludes more volume - RNAP) • - concentration gradient formed inside the brush (influenced by the position, direction, and TX unit length) • The decrease in TX rate in DNA brush after 1 h remains unexplained • RNA accumulation in the brush could be the reason behind slowing down of the TX reaction • Research opens further question of whether compartmentalization and gene expression in living cells are affected by similar parameters conditions • Offers ideas for barrier-free construction of synthetic cells Fig. 1. (A) Scheme of synthetic DNA brush (2,160 bp) with a ∼300 bp TX unit in between T7 promoter and terminator (facing IN). (B) Scheme of 6 brush arrangements with TX units (Red). Fig. 3. (A) DNA densities of the individual DNA brushes. (B) TX rate as a function of coding DNA density for mixed (empty/filled blue circles) and coding-only (empty/filled red squares) DNA brushes.