Antti Häkkinen

Delayed Stochastic Model of Transcription at the Single Nucleotide Level

We present a delayed stochastic model of transcription at the single nucleotide level. The model accounts for the promoter open complex formation and includes alternative pathways to elongation, namely pausing, arrest, misincorporation and editing, pyrophosphorolysis, and premature termination. We confront the dynamics of this detailed model with a single-step multi-delayed stochastic model and with measurements of expression of a repressed gene at the single molecule level. At low expression rates both models match the experiments but, at higher rates the two models differ significantly, with consequences to cell-to-cell phenotypic variability. The alternative pathway reactions, due to, for example, causing polymerases to collide more often on the template, are the cause for the difference in dynamical behaviors. Next, we confront the model with measurements of the transcriptional dynamics at the single RNA level of an induced gene and show that RNA production, besides its bursting dynamics, also exhibits pulses (2 or more RNAs produced in intervals smaller than the smallest interval between initiations). The distribution of occurrences and amplitudes of pulses match the experimental measurements. This pulsing and the noise at the elongation stage are shown to play a role in the dynamics of a genetic switch.

In vivo kinetics of transcription initiation of the lar promoter in Escherichia coli. Evidence for a sequential mechanism with two rate-limiting steps

BackgroundIn Escherichia coli the mean and cell-to-cell diversity in RNA numbers of different genes vary widely. This is likely due to different kinetics of transcription initiation, a complex process with multiple rate-limiting steps that affect RNA production.ResultsWe measured the in vivo kinetics of production of individual RNA molecules under the control of the lar promoter in E. coli. From the analysis of the distributions of intervals between transcription events in the regimes of weak and medium induction, we find that the process of transcription initiation of this promoter involves a sequential mechanism with two main rate-limiting steps, each lasting hundreds of seconds. Both steps become faster with increasing induction by IPTG and Arabinose.ConclusionsThe two rate-limiting steps in initiation are found to be important regulators of the dynamics of RNA production under the control of the lar promoter in the regimes of weak and medium induction. Variability in the intervals between consecutive RNA productions is much lower than if there was only one rate-limiting step with a duration following an exponential distribution. The methodology proposed here to analyze the in vivo dynamics of transcription may be applicable at a genome-wide scale and provide valuable insight into the dynamics of prokaryotic genetic networks.

Single-molecule dynamics of transcription of the lar promoter

We measured the in vivo production of RNA molecules tagged with MS2d-GFP in Escherichia coli, driven by the lar promoter, under weak and medium induction. The distributions of intervals between consecutive productions of RNAs are found to be sub-exponential, and the process of RNA production is found to be sub-Poissonian. We discuss possible models of transcription initiation and, based on our results and previous in vitro measurements, find that a sequential two-step model of transcription initiation at the promoter region explains the results well.

Asymmetric disposal of individual protein aggregates in Escherichia coli, one aggregate at a time

Escherichia coli cells employ an asymmetric strategy at division, segregating unwanted substances to older poles, which has been associated with aging in these organisms. The kinetics of this process is still poorly understood. Using the MS2 coat protein fused to green fluorescent protein (GFP) and a reporter construct with multiple MS2 binding sites, we tracked individual RNA-MS2-GFP complexes in E. coli cells from the time when they were produced. Analyses of the kinetics and brightness of the spots showed that these spots appear in the midcell region, are composed of a single RNA-MS2-GFP complex, and reach a pole before another target RNA is formed, typically remaining there thereafter. The choice of pole is probabilistic and heavily biased toward one pole, similar to what was observed by previous studies regarding protein aggregates. Additionally, this mechanism was found to act independently on each disposed molecule. Finally, while the RNA-MS2-GFP complexes were disposed of, the MS2-GFP tagging molecules alone were not. We conclude that this asymmetric mechanism to segregate damage at the expense of aging individuals acts probabilistically on individual molecules and is capable of the accurate classification of molecules for disposal.

NF-Y influences directionality of transcription from the bidirectional Mrps12/Sarsm promoter in both mouse and human cells.

The bidirectional mammalian promoter for mitoribosomal protein S12 (Mrps12) and mitochondrial seryl-tRNA ligase (Sarsm) contains an array of four CCAAT boxes separated by 34-49 bp. In mouse, these elements were shown previously to interact with transcription factor NF-Y and to be required for efficient transcription. Here we show that the CCAAT boxes of the human promoter also influence relative transcriptional activities in the two directions, although they are not absolutely required for transcription. By mutating CCAAT boxes in all possible permutations, we demonstrate that their function is combinatorial, although not simply additive. EMSA indicated that NF-Y interacts with the array in two alternate ways related to the directional selectivity of transcription. Inversion and/or exchange of individual CCAAT boxes had minimal effects on directional selectivity. Over-expression of wild-type or dominant-negative NF-Y affected transcription in the Sarsm direction only, but in human cells, concomitant expression of dominant-negative constructs for other factors was needed to reveal such effects. We propose that the array of NF-Y type CCAAT boxes maintains bidirectional transcription with an appropriate directional selectivity. Computational analysis confirmed that NF-Y type CCAAT boxes are found preferentially in bidirectional promoters, but many such promoters lack them and must be regulated in another way.

Genome wide study of NF-Y type CCAAT boxes in unidirectional and bidirectional promoters in human and mouse

A subset of CCAAT boxes is known as binding sites for the transcription factor NF-Y. We characterize their number, mismatches to the consensus sequence, and locations in bidirectional and unidirectional promoter sequences in human and mouse. We confront the findings with an analytical null model of DNA sequences and find that NF-Y type CCAAT boxes play key, but distinct roles in the two types of promoters. They are found above chance in both, but in unidirectional only when having few mismatches. In bidirectional, the relative positions of multiple boxes differ from what is expected by chance, suggesting the need for contiguity. In agreement, when there are four boxes (four-box configurations), these have much lower number of mismatches than expected in bidirectional promoters alone. Positioning of the first box differs in the two types of promoters and the null model, and mismatches and positioning are found to be correlated. Finally, four-box configurations are conserved between human and mouse, supporting the relevance of the findings. We conclude that bidirectional and unidirectional promoters, while sharing some similarities, appear to possess distinct regulatory mechanisms at the sequence level.

Dissecting the stochastic transcription initiation process in live Escherichia coli

We investigate the hypothesis that, in Escherichia coli, while the concentration of RNA polymerases differs in different growth conditions, the fraction of RNA polymerases free for transcription remains approximately constant within a certain range of these conditions. After establishing this, we apply a standard model-fitting procedure to fully characterize the in vivo kinetics of the rate-limiting steps in transcription initiation of the Plac/ara-1 promoter from distributions of intervals between transcription events in cells with different RNA polymerase concentrations. We find that, under full induction, the closed complex lasts ∼788 s while subsequent steps last ∼193 s, on average. We then establish that the closed complex formation usually occurs multiple times prior to each successful initiation event. Furthermore, the promoter intermittently switches to an inactive state that, on average, lasts ∼87 s. This is shown to arise from the intermittent repression of the promoter by LacI. The methods employed here should be of use to resolve the rate-limiting steps governing the in vivo dynamics of initiation of prokaryotic promoters, similar to established steady-state assays to resolve the in vitro dynamics.

Research article: Dynamical effects of transcriptional pause-prone sites

We study how long pause-prone sites, commonly sequence-dependent, affect transcription and RNA temporal levels in a delayed stochastic model of transcription at the single nucleotide level. We vary pause propensity, duration and the probability of premature termination of elongation at the pause site. We also study the effects of multiple pause sites. We show that pause sites can be used to fine-tune noise strength and burst size distribution of RNA levels. Varying pause rate and duration alone affects bursting but noise is not significantly affected. Noise strength can be changed by varying both parameters and, even more pronouncedly, by varying the probability of premature termination. Adding multiple pause sites amplifies the increase in noise and bursting. This regulatory mechanism of noise and bursting, being evolvable, may partially explain how different genes exhibit a wide spectrum of different behaviors. The results might assist the engineering of genes with a desired degree of noise.

Increased cytoplasm viscosity hampers aggregate polar segregation in Escherichia coli

In Escherichia coli, under optimal conditions, protein aggregates associated with cellular aging are excluded from midcell by the nucleoid. We study the functionality of this process under sub‐optimal temperatures from population and time lapse images of individual cells and aggregates and nucleoids within. We show that, as temperature decreases, aggregates become homogeneously distributed and uncorrelated with nucleoid size and location. We present evidence that this is due to increased cytoplasm viscosity, which weakens the anisotropy in aggregate displacements at the nucleoid borders that is responsible for their preference for polar localisation. Next, we show that in plasmolysed cells, which have increased cytoplasm viscosity, aggregates are also not preferentially located at the poles. Finally, we show that the inability of cells with increased viscosity to exclude aggregates from midcell results in enhanced aggregate concentration in between the nucleoids in cells close to dividing. This weakens the asymmetries in aggregate numbers between sister cells of subsequent generations required for rejuvenating cell lineages. We conclude that the process of exclusion of protein aggregates from midcell is not immune to stress conditions affecting the cytoplasm viscosity. The findings contribute to our understanding of E. colis internal organisation and functioning, and its fragility to stressful conditions.

Effects of Rate-Limiting Steps in Transcription Initiation on Genetic Filter Motifs

The behavior of genetic motifs is determined not only by the gene-gene interactions, but also by the expression patterns of the constituent genes. Live single-molecule measurements have provided evidence that transcription initiation is a sequential process, whose kinetics plays a key role in the dynamics of mRNA and protein numbers. The extent to which it affects the behavior of cellular motifs is unknown. Here, we examine how the kinetics of transcription initiation affects the behavior of motifs performing filtering in amplitude and frequency domain. We find that the performance of each filter is degraded as transcript levels are lowered. This effect can be reduced by having a transcription process with more steps. In addition, we show that the kinetics of the stepwise transcription initiation process affects features such as filter cutoffs. These results constitute an assessment of the range of behaviors of genetic motifs as a function of the kinetics of transcription initiation, and thus will aid in tuning of synthetic motifs to attain specific characteristics without affecting their protein products.