Das, N. ; Valjavec-Gratian, M. ; Basuray, A. N. ; Fekete, R. A. ; Papp, P. P. ; Paulsson, J. ; Chattoraj, D. K. Multiple homeostatic mechanisms in the control of P1 plasmid replication. Proceedings of the National Academy of Sciences of USA 2005,
102, 2856-61.
AbstractMany organisms control initiation of DNA replication by limiting supply or activity of initiator proteins. In plasmids, such as P1, initiators are limited primarily by transcription and dimerization. However, the relevance of initiator limitation to plasmid copy number control has appeared doubtful, because initiator oversupply increases the copy number only marginally. Copy number control instead has been attributed to initiator-mediated plasmid pairing ("handcuffing"), because initiator mutations to handcuffing deficiency elevates the copy number significantly. Here, we present genetic evidence of a role for initiator limitation in plasmid copy number control by showing that autorepression-defective initiator mutants also can elevate the plasmid copy number. We further show, by quantitative modeling, that initiator dimerization is a homeostatic mechanism that dampens active monomer increase when the protein is oversupplied. This finding implies that oversupplied initiator proteins are largely dimeric, partly accounting for their limited ability to increase copy number. A combination of autorepression, dimerization, and handcuffing appears to account fully for control of P1 plasmid copy number.
Golding, I. ; Paulsson, J. ; Zawilski, S. M. ; Cox, E. C. Real-time kinetics of gene activity in individual bacteria. Cell 2005,
123, 1025-36.
AbstractProtein levels have been shown to vary substantially between individual cells in clonal populations. In prokaryotes, the contribution to such fluctuations from the inherent randomness of gene expression has largely been attributed to having just a few transcripts of the corresponding mRNAs. By contrast, eukaryotic studies tend to emphasize chromatin remodeling and burst-like transcription. Here, we study single-cell transcription in Escherichia coli by measuring mRNA levels in individual living cells. The results directly demonstrate transcriptional bursting, similar to that indirectly inferred for eukaryotes. We also measure mRNA partitioning at cell division and correlate mRNA and protein levels in single cells. Partitioning is approximately binomial, and mRNA-protein correlations are weaker earlier in the cell cycle, where cell division has recently randomized the relative concentrations. Our methods further extend protein-based approaches by counting the integer-valued number of transcript with single-molecule resolution. This greatly facilitates kinetic interpretations in terms of the integer-valued random processes that produce the fluctuations.