The spatial biology of transcription and translation in rapidly growing Escherichia coli.

Single-molecule fluorescence provides high resolution spatial distributions of ribosomes and RNA polymerase (RNAP) in live, rapidly growing Escherichia coli. Ribosomes are more strongly segregated from the nucleoids (chromosomal DNA) than previous widefield fluorescence studies suggested. While most transcription may be co-translational, the evidence indicates that most translation occurs on free mRNA copies that have diffused from the nucleoids to a ribosome-rich region. Analysis of time-resolved images of the nucleoid spatial distribution after treatment with the transcription-halting drug rifampicin and the translation-halting drug chloramphenicol shows that both drugs cause nucleoid contraction on the 0-3 min timescale. This is consistent with the transertion hypothesis. We suggest that the longer-term (20-30 min) nucleoid expansion after Rif treatment arises from conversion of 70S-polysomes to 30S and 50S subunits, which readily penetrate the nucleoids. Monte Carlo simulations of a polymer bead model built to mimic the chromosomal DNA and ribosomes (either 70S-polysomes or 30S and 50S subunits) explain spatial segregation or mixing of ribosomes and nucleoids in terms of excluded volume and entropic effects alone. A comprehensive model of the transcription-translation-transertion system incorporates this new information about the spatial organization of the E. coli cytoplasm. We propose that transertion, which radially expands the nucleoids, is essential for recycling of 30S and 50S subunits from ribosome-rich regions back into the nucleoids. There they initiate co-transcriptional translation, which is an important mechanism for maintaining RNAP forward progress and protecting the nascent mRNA chain. Segregation of 70S-polysomes from the nucleoid may facilitate rapid growth by shortening the search time for ribosomes to find free mRNA concentrated outside the nucleoid and the search time for RNAP concentrated within the nucleoid to find transcription initiation sites

Electronic spectroscopy of jet-cooled combustion radicals. Final report

Jet-cooled spectra of 1-methylvinoxy and 2-methylvinoxy are reported and analyzed with help from high-level electronic structure calculations

Modified Pearson correlation coefficient for two-color imaging in spherocylindrical cells

Abstract The revolution in fluorescence microscopy enables sub-diffraction-limit (“superresolution”) localization of hundreds or thousands of copies of two differently labeled proteins in the same live cell. In typical experiments, fluorescence from the entire three-dimensional (3D) cell body is projected along the z-axis of the microscope to form a 2D image at the camera plane. For imaging of two different species, here denoted “red” and “green”, a significant biological question is the extent to which the red and green spatial distributions are positively correlated, anti-correlated, or uncorrelated. A commonly used statistic for assessing the degree of linear correlation between two image matrices R and G is the Pearson Correlation Coefficient (PCC). PCC should vary from − 1 (perfect anti-correlation) to 0 (no linear correlation) to + 1 (perfect positive correlation). However, in the special case of spherocylindrical bacterial cells such as E. coli or B. subtilis, we show that the PCC fails both qualitatively and quantitatively. PCC returns the same + 1 value for 2D projections of distributions that are either perfectly correlated in 3D or completely uncorrelated in 3D. The PCC also systematically underestimates the degree of anti-correlation between the projections of two perfectly anti-correlated 3D distributions. The problem is that the projection of a random spatial distribution within the 3D spherocylinder is non-random in 2D, whereas PCC compares every matrix element of R or G with the constant mean value R¯ $$\overline{R}$$ or G¯ $$\overline{G}$$. We propose a modified Pearson Correlation Coefficient (MPCC) that corrects this problem for spherocylindrical cell geometry by using the proper reference matrix for comparison with R and G. Correct behavior of MPCC is confirmed for a variety of numerical simulations and on experimental distributions of HU and RNA polymerase in live E. coli cells. The MPCC concept should be generalizable to other cell shapes

Isotope, Electric Field, and Vibrational State Dependence of Single Rotational Level Lifetimes of S1 Formaldehyde

Additional single rovibronic level lifetimes of S{sub 1} H{sub 2}CO and D{sub 2}CO have been measured under collisionless conditions. The H{sub 2}CO 4{sup 1} lifetimes vary at least a factor of 150, from 20 nsec to 3.10 {micro}sec. The observed D{sub 2}CO 4{sup 1} lifetimes fluctuate about {+-} 20% around a mean value of 6.2 {micro}sec, which is probably close to the pure radiative lifetime. In contrast, the observed D{sub 2}CO 4{sup 3} lifetimes vary from 1.09 to 2.46 {micro}sec and the 2{sup 1} 4{sup 3} lifetimes vary from 212 nsec to 1.61 {micro}sec. The onset of rotational state lifetime fluctuations in D{sub 2}CO thus coincides with the high pressure D{sub 2} + CO photochemical threshold. All of these results are explained in terms of a collisionless sequential decay mechanism, S{sub 1} {yields} S{sub 0} {yields} H{sub 2}(D{sub 2}) + CO. The last step probably involves tunneling through a barrier for the lower energies studied. For several H{sub 2}CO 4{sup 1} rotational levels application of a uniform external electric field of 0-4.6 kV/cm can change the fluorescence lifetime by at least a factor of 4. This result is understood in terms of small ({approx}< 0.05 cm{sup -1}) shifts in S{sub 1}-S{sub 0} energy spacings. Quantitative estimates of S{sub 1}-S{sub 0} intramolecular couplings, S{sub 0} widths due to dissociation, and S{sub 0} level spacings are derived in favorable cases