Salerno's model of DNA reanalysed: could solitons have biological significance?

We investigate the sequence-dependent behaviour of localised excitations in a toy, nonlinear model of DNA base-pair opening originally proposed by Salerno. Specifically we ask whether ``breather'' solitons could play a role in the facilitated location of promoters by RNA polymerase. In an effective potential formalism, we find excellent correlation between potential minima and {\em Escherichia coli} promoter recognition sites in the T7 bacteriophage genome. Evidence for a similar relationship between phage promoters and downstream coding regions is found and alternative reasons for links between AT richness and transcriptionally-significant sites are discussed. Consideration of the soliton energy of translocation provides a novel dynamical picture of sliding: steep potential gradients correspond to deterministic motion, while ``flat'' regions, corresponding to homogeneous AT or GC content, are governed by random, thermal motion. Finally we demonstrate an interesting equivalence between planar, breather solitons and the helical motion of a sliding protein ``particle'' about a bent DNA axis.Comment: Latex file 20 pages, 5 figures. Manuscript of paper to appear in J. Biol. Phys., accepted 02/09/0

Stationary phase induction of RpoS in enteric bacteria

In enteric bacteria, stress adaptation is mediated by the RpoS protein, one of several sigma-factors that, in association with RNA polymerase, collectively allow a tailored transcriptional response to environmental cues. Stress stimuli including low temperature, osmotic shock, and starvation all result in a substantial increase in RpoS abundance. Perhaps the most pronounced affect is observed during growth to stationary phase (SP) in rich medium. The mechanism of regulation depends on the specific signal, but may occur at the level of transcription, translation, protein activity or targeted proteolysis. In both Escherichia coli and Salmonella enterica cultured in rich undefined medium, the RpoS protein is barely detectable during exponential growth and increases \u3e30-fold as cells enter SP. Under these conditions, SP induction depends on transcriptional and translational control with proteolysis affecting basal levels but not regulation per se. The transiently expressed Fis protein, whose abundance inversely correlates to that of RpoS, binds just upstream of the primary rpoS promoter and represses transcription nearly 10-fold specifically during exponential growth. SP induction at the translational level relies on a novel form of genetic control dependent on the 24 nucleotides preceding the rpoS initiation codon (ribosome-binding sequence, RBS). The RNA secondary structure of the rpoS RBS is necessary and sufficient for a nearly 10-fold translational increase during SP. Control at this level is not a result of differential transcript stability, nor does it involve the known rpoS regulators ppGpp, DksA, HU, Hfq or the small regulatory RNAs, DsrA and RprA. The environmental stimuli that trigger RBS-mediated SP induction of rpoS translation also remain unknown, but similar to transcriptional control, regulation is only seen in rich undefined media. Collectively, transcriptional repression by Fis and RBS-mediated induction at the translational level account for approximately 95% of the overall SP induction of RpoS

Stochasticity and traffic jams in the transcription of ribosomal RNA: Intriguing role of termination and antitermination

In fast growing bacteria, ribosomal RNA (rRNA) is required to be transcribed at very high rates to sustain the high cellular demand on ribosome synthesis. This results in dense traffic of RNA polymerases (RNAP). We developed a stochastic model, integrating results of single-molecule and quantitative in vivo studies of E. coli, to evaluate the quantitative effect of pausing, termination, and antitermination on rRNA transcription. Our calculations reveal that in dense RNAP traffic, spontaneous pausing of RNAP can lead to severe "traffic jams", as manifested in the broad distribution of inter-RNAP distances and can be a major factor limiting transcription and hence growth. Our results suggest the suppression of these pauses by the ribosomal antitermination complex to be essential at fast growth. Moreover, unsuppressed pausing by even a few non-antiterminated RNAPs can already reduce transcription drastically under dense traffic. However, the termination factor Rho can remove the non-antiterminated RNAPs and restore fast transcription. The results thus suggest an intriguing role by Rho to enhance rather than attenuate rRNA transcription.Comment: includes Supporting Informatio

Rate-limiting Steps in Transcription Initiation are Key Regulatory Mechanisms of Escherichia coli Gene Expression Dynamics

In all living organisms, the “blueprints of life” are documented in the genetic material. This material is composed of genes, which are regions of DNA coding for proteins. To produce proteins, cells read the information on the DNA with the help of molecular machines, such as RNAp holoenzymes and a factors. Proteins carry out the cellular functions required for survival and, as such, cells deal with challenging environments by adjusting their gene expression pattern. For this, cells constantly perform decision- making processes of whether or not to actively express a protein, based on intracellular and environmental cues. In Escherichia coli, gene expression is mostly regulated at the stage of transcription initiation. Although most of its regulatory molecules have been identified, the dynamics and regulation of this step remain elusive. Due to a limited number of specific regulatory molecules in the cells, the stochastic fluctuations of these molecular numbers can result in a sizeable temporal change in the numbers of transcription outputs (RNA and proteins) and have consequences on the phenotype of the cells. To understand the dynamics of this process, one should study the activity of the gene by tracking mRNA and protein production events at a detailed level. Recent advancements in single-molecule detection techniques have been used to image and track individually labeled fluorescent macromolecules of living cells. This allows investigating the intermolecular dynamics under any given condition. In this thesis, by using in vivo, single-RNA time-lapse microscopy techniques along with stochastic modelling techniques, we studied the kinetics of multi-rate limiting steps in the transcription process of multiple promoters, in various conditions. Specifically, first, we established a novel method of dissecting transcription in Escherichia coli that combines state-of-the-art microscopy measurements and model fitting techniques to construct detailed models of the rate-limiting steps governing the in vivo transcription initiation of a synthetic Lac-ara-1 promoter. After that, we estimated the duration of the closed and open complex formation, accounting for the rate of reversibility of the first step. From this, we also estimated the duration of periods of promoter inactivity, from which we were able to determine the contribution from each step to the distribution of intervals between consecutive RNA productions in individual cells. Second, using the above method, we studied the a factor selective mechanisms for indirect regulation of promoters whose transcription is primarily initiated by RNAp holoenzymes carrying a70. From the analysis, we concluded that, in E. coli, a promoter’s responsiveness to indirect regulation by a factor competition is determined by its sequence-dependent, dynamically regulated multi-step initiation kinetics. Third, we investigated the effects of extrinsic noise, arising from cell-to-cell variability in cellular components, on the single-cell distribution of RNA numbers, in the context of cell lineages. For this, first, we used stochastic models to predict the variability in the numbers of molecules involved in upstream processes. The models account for the intake of inducers from the environment, which acts as a transient source of variability in RNA production numbers, as well as for the variability in the numbers of molecular species controlling transcription of an active promoter, which acts as a constant source of variability in RNA numbers. From measurement analysis, we demonstrated the existence of lineage-to-lineage variability in gene activation times and mean transcription rates. Finally, we provided evidence that this can be explained by differences in the kinetics of the rate-limiting steps in transcription and of the induction scheme, from which it is possible to conclude that these variabilities differ between promoters and inducers used. Finally, we studied how the multi-rate limiting steps in the transcription initiation are capable of tuning the asymmetry and tailedness of the distribution of time intervals between consecutive RNA production events in individual cells. For this, first, we considered a stochastic model of transcription initiation and predicted that the asymmetry and tailedness in the distribution of intervals between consecutive RNA production events can differ by tuning the rate-limiting steps in transcription. Second, we validated the model with measurements from single-molecule RNA microscopy of transcription kinetics of multiple promoters in multiple conditions. Finally, from our results, we concluded that the skewness and kurtosis in RNA and protein production kinetics are subject to regulation by the kinetics of the steps in transcription initiation and affect the single-cell distributions of RNAs and, thus, proteins. We further showed that this regulation can significantly affect the probability of RNA and protein numbers to cross specific thresholds. Overall, the studies conducted in this thesis are expected to contribute to a better understanding of the dynamic process of bacterial gene expression. The advanced data and image analysis techniques and novel stochastic modeling approaches that we developed during the course of these studies, will allow studying in detail the in vivo regulation of multi-rate limiting steps of transcription initiation of any given promoter. In addition, by tuning the kinetics of the rate-limiting steps in the transcription initiation as executed here should allow engineering new promoters, with predefined RNA and, thus, protein production dynamics in Escherichia coli

Transcription Initiation Studies With Bacillus Subtilis Promoters Containing Curved DNA.

Many promoters contain a curved DNA component essential for high levels of transcription activity. The present work approached the study of these unusual DNA structures in a twofold manner. First, promoter binding by RNA polymerase from Bacillus subtilis and Escherichia coli was examined using a collection of promoters containing DNA curvature. Promoter binding by both RNA polymerases was governed primarily by the nucleotide sequence at the highly conserved $-$10 and $-$35 regions of the promoters. However, the presence of curved DNA, immediately upstream of the $-$35 region of the promoter, predictably increased binding to those promoters which contained curved DNA by the B. subtilis RNA polymerase. Binding by the E. coli RNA polymerase was modestly affected by DNA curvature. The second approach was to characterize the mechanism involved in transcription stimulation by this curve or intrinsically bent DNA. Generally, the proposed mechanisms for transcription initiation include enzyme conformational changes leading to strand separation. Formation of RNA polymerase-promoter complexes that entail significant conformational changes is sensitive to changes in temperature. A goal in the second part of this work was to test if curved DNA influenced a step in transcription initiation that was sensitive to temperature changes. The formation of open promoter complexes was measured using a B. subtilis phage promoter containing curved DNA upstream of the $-$35 region, the Alu156 promoter, and mutants of Alu156 in which the curved DNA sequences were displaced upstream. Open promoter complexes were measured by (1) a run-off transcription assay limited to a single round of initiation and (2) the direct detection of single stranded DNA using potassium permanganate cleavage. Promoters with properly aligned curved DNA formed open promoter complexes at lower temperatures than promoters with misaligned curved DNA. Also, curved DNA enhanced formation of open promoter complexes as measured by actual strand separation. A model describing the effect of curved DNA on open promoter complexes was proposed

Nucleic Acids Res.

A remarkable feature of many small non-coding RNAs (sRNAs) of Escherichia coli and Salmonella is their accumulation in the stationary phase of bacterial growth. Several stress response regulators and sigma factors have been reported to direct the transcription of stationary phase-specific sRNAs, but a widely conserved sRNA gene that is controlled by the major stationary phase and stress sigma factor, Sigma(S) (RpoS), has remained elusive. We have studied in Salmonella the conserved SdsR sRNA, previously known as RyeB, one of the most abundant stationary phase-specific sRNAs in E. coli. Alignments of the sdsR promoter region and genetic analysis strongly suggest that this sRNA gene is selectively transcribed by Sigma(S). We show that SdsR down-regulates the synthesis of the major Salmonella porin OmpD by Hfq-dependent base pairing; SdsR thus represents the fourth sRNA to regulate this major outer membrane porin. Similar to the InvR, MicC and RybB sRNAs, SdsR recognizes the ompD mRNA in the coding sequence, suggesting that this mRNA may be primarily targeted downstream of the start codon. The SdsR-binding site in ompD was localized by 3'-RACE, an experimental approach that promises to be of use in predicting other sRNA-target interactions in bacteria

Conserved and Variable Functions of the σ(E) Stress Response in Related Genomes

Bacteria often cope with environmental stress by inducing alternative sigma (σ) factors, which direct RNA polymerase to specific promoters, thereby inducing a set of genes called a regulon to combat the stress. To understand the conserved and organism-specific functions of each σ, it is necessary to be able to predict their promoters, so that their regulons can be followed across species. However, the variability of promoter sequences and motif spacing makes their prediction difficult. We developed and validated an accurate promoter prediction model for Escherichia coli σ(E), which enabled us to predict a total of 89 unique σ(E)-controlled transcription units in E. coli K-12 and eight related genomes. σ(E) controls the envelope stress response in E. coli K-12. The portion of the regulon conserved across genomes is functionally coherent, ensuring the synthesis, assembly, and homeostasis of lipopolysaccharide and outer membrane porins, the key constituents of the outer membrane of Gram-negative bacteria. The larger variable portion is predicted to perform pathogenesis-associated functions, suggesting that σ(E) provides organism-specific functions necessary for optimal host interaction. The success of our promoter prediction model for σ(E) suggests that it will be applicable for the prediction of promoter elements for many alternative σ factors