CarD and RbpA modify the kinetics of initial transcription and slow promoter escape of the Mycobacterium tuberculosis RNA polymerase.

The pathogen Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, enacts unique transcriptional regulatory mechanisms when subjected to host-derived stresses. Initiation of transcription by the Mycobacterial RNA polymerase (RNAP) has previously been shown to exhibit different open complex kinetics and stabilities relative to Escherichia coli (Eco) RNAP. However, transcription initiation rates also depend on the kinetics following open complex formation such as initial nucleotide incorporation and subsequent promoter escape. Here, using a real-time fluorescence assay, we present the first in-depth kinetic analysis of initial transcription and promoter escape for the Mtb RNAP. We show that in relation to Eco RNAP, Mtb displays slower initial nucleotide incorporation but faster overall promoter escape kinetics on the Mtb rrnAP3 promoter. Furthermore, in the context of the essential transcription factors CarD and RbpA, Mtb promoter escape is slowed via differential effects on initially transcribing complexes. Finally, based on their ability to increase the rate of open complex formation and decrease the rate of promoter escape, we suggest that CarD and RbpA are capable of activation or repression depending on the rate-limiting step of a given promoter's basal initiation kinetics.

Cooperative stabilization of Mycobacterium tuberculosis rrnAP3 promoter open complexes by RbpA and CarD.

The essential mycobacterial transcriptional regulators RbpA and CarD act to modulate transcription by associating to the initiation complex and increasing the flux of transcript production. Each of these factors interacts directly with the promoter DNA template and with RNA polymerase (RNAP) holoenzyme. We recently reported on the energetics of CarD-mediated open complex stabilization on the Mycobacterium tuberculosis rrnAP3 ribosomal promoter using a stopped-flow fluorescence assay. Here, we apply this approach to RbpA and show that RbpA stabilizes RNAP-promoter open complexes (RPo) via a distinct mechanism from that of CarD. Furthermore, concentration-dependent stopped-flow experiments with both factors reveal positive linkage (cooperativity) between RbpA and CarD with regard to their ability to stabilize RPo The observation of positive linkage between RbpA and CarD demonstrates that the two factors can act on the same transcription initiation complex simultaneously. Lastly, with both factors present, the kinetics of open complex formation is significantly faster than in the presence of either factor alone and approaches that of E. coli RNAP on the same promoter. This work provides a quantitative framework for the molecular mechanisms of these two essential transcription factors and the critical roles they play in the biology and pathology of mycobacteria.

Effects of Increasing the Affinity of CarD for RNA Polymerase on Mycobacterium tuberculosis Growth, rRNA Transcription, and Virulence.

CarD is an essential RNA polymerase (RNAP) interacting protein in Mycobacterium tuberculosis that stimulates formation of RNAP-promoter open complexes. CarD plays a complex role in M. tuberculosis growth and virulence that is not fully understood. Therefore, to gain further insight into the role of CarD in M. tuberculosis growth and virulence, we determined the effect of increasing the affinity of CarD for RNAP. Using site-directed mutagenesis guided by crystal structures of CarD bound to RNAP, we identified amino acid substitutions that increase the affinity of CarD for RNAP. Using these substitutions, we show that increasing the affinity of CarD for RNAP increases the stability of the CarD protein in M. tuberculosis In addition, we show that increasing the affinity of CarD for RNAP increases the growth rate in M. tuberculosis without affecting 16S rRNA levels. We further show that increasing the affinity of CarD for RNAP reduces M. tuberculosis virulence in a mouse model of infection despite the improved growth rate in vitro Our findings suggest that the CarD-RNAP interaction protects CarD from proteolytic degradation in M. tuberculosis, establish that growth rate and rRNA levels can be uncoupled in M. tuberculosis and demonstrate that the strength of the CarD-RNAP interaction has been finely tuned to optimize virulence. IMPORTANCE: Mycobacterium tuberculosis, the causative agent of tuberculosis, remains a major global health problem. In order to develop new strategies to battle this pathogen, we must gain a better understanding of the molecular processes involved in its survival and pathogenesis. We have previously identified CarD as an essential transcriptional regulator in mycobacteria. In this study, we detail the effects of increasing the affinity of CarD for RNAP on transcriptional regulation, CarD protein stability, and virulence. These studies expand our understanding of the global transcription regulator CarD, provide insight into how CarD activity is regulated, and broaden our understanding of prokaryotic transcription.

CarD stabilizes mycobacterial open complexes via a two-tiered kinetic mechanism.

CarD is an essential and global transcriptional regulator in mycobacteria. While its biological role is unclear, CarD functions by interacting directly with RNA polymerase (RNAP) holoenzyme promoter complexes. Here, using a fluorescent reporter of open complex, we quantitate RPo formation in real time and show that Mycobacterium tuberculosis CarD has a dramatic effect on the energetics of RNAP bound complexes on the M. tuberculosis rrnAP3 ribosomal RNA promoter. The data reveal that Mycobacterium bovis RNAP exhibits an unstable RPo that is stabilized by CarD and suggest that CarD uses a two-tiered, concentration-dependent mechanism by associating with open and closed complexes with different affinities. Specifically, the kinetics of open-complex formation can be explained by a model where, at saturating concentrations of CarD, the rate of bubble collapse is slowed and the rate of opening is accelerated. The kinetics and open-complex stabilities of CarD mutants further clarify the roles played by the key residues W85, K90 and R25 previously shown to affect CarD-dependent gene regulation in vivo. In contrast to M. bovis RNAP, Escherichia coli RNAP efficiently forms RPo on rrnAP3, suggesting an important difference between the polymerases themselves and highlighting how transcriptional machinery can vary across bacterial genera.

A Kinetic Signature for Parallel Pathways: Conformational Selection and Induced Fit. Links and Disconnects between Observed Relaxation Rates and Fractional Equilibrium Flux under Pseudo-First-Order Conditions.

Molecular association plays a ubiquitous role in biochemistry and is often accompanied by conformational exchange in one or both binding partners. Traditionally, two limiting mechanisms are considered for the association of two molecules. In a conformational selection (CS) mechanism, a ligand preferentially binds to a subset of conformations in its binding partner. In contrast, an induced fit (IF) mechanism describes the ligand-dependent isomerization of the binding partner in which binding occurs prior to conformational exchange. Measurements of the ligand concentration dependence of observed rates of relaxation are commonly used to probe whether CS or IF is taking place. Here we consider a four-state thermodynamic cycle subject to detailed balance and demonstrate the existence of a relatively unexplored class of kinetic signatures where an initial decrease in the observed rate is followed by a subsequent increase under pseudo-first-order conditions. We elucidate regions of rate space necessary to generate a nonmonotonic observed rate and show that, under certain conditions, the position of the minimum of the observed rate correlates with a transition in equilibrium flux between CS and IF pathways. Furthermore, we demonstrate that monotonic trends in the observed rate can occur when both CS and IF mechanisms are taking place, suggesting that caution must be taken not to overinterpret monotonic trends as evidence of the absence of either CS or IF. Lastly, we conclude that a nonmonotonic kinetic signature is uniquely unambiguous in the sense that when this trend is observed, one may conclude that both CS and IF mechanistic paths are utilized.

Oxysterols are allosteric activators of the oncoprotein Smoothened.

Oxysterols are a class of endogenous signaling molecules that can activate the Hedgehog pathway, which has critical roles in development, regeneration and cancer. However, it has been unclear how oxysterols influence Hedgehog signaling, including whether their effects are mediated through a protein target or indirectly through effects on membrane properties. To answer this question, we synthesized the enantiomer and an epimer of the most potent oxysterol, 20(S)-hydroxycholesterol. Using these molecules, we show that the effects of oxysterols on Hedgehog signaling are exquisitely stereoselective, consistent with the hypothesis that they function through a specific protein target. We present several lines of evidence that this protein target is the seven-pass transmembrane protein Smoothened, a major drug target in oncology. Our work suggests that these enigmatic sterols, which have multiple effects on cell physiology, may act as ligands for signaling receptors and provides a generally applicable framework for probing sterol signaling mechanisms.

Nearly maximal information gain due to time integration in central dogma reactions.

Living cells process information about their environment through the central dogma processes of transcription and translation, which drive the cellular response to stimuli. Here, we study the transfer of information from environmental input to the transcript and protein expression levels. Evaluation of both experimental and analogous simulation data reveals that transcription and translation are not two simple information channels connected in series. Instead, we demonstrate that the central dogma reactions often create a time-integrating information channel, where the translation channel receives and integrates multiple outputs from the transcription channel. This information channel model of the central dogma provides new information-theoretic selection criteria for the central dogma rate constants. Using the data for four well-studied species we show that their central dogma rate constants achieve information gain because of time integration while also keeping the loss because of stochasticity in translation relatively low (<0.5 bits).

Single-cell measurement quality in bits.

Single-cell measurements have revolutionized our understanding of heterogeneity in cellular response. However, there is no universally comparable way to assess single-cell measurement quality. Here, we show how information theory can be used to assess and compare single-cell measurement quality in bits, which provides a universally comparable metric for information content. We anticipate that the experimental and theoretical approaches we show here will generally enable comparisons of quality between any single-cell measurement methods.

Comparison of bias and resolvability in single-cell and single-transcript methods.

Single-cell and single-transcript measurement methods have elevated our ability to understand and engineer biological systems. However, defining and comparing performance between methods remains a challenge, in part due to the confounding effects of experimental variability. Here, we propose a generalizable framework for performing multiple methods in parallel using split samples, so that experimental variability is shared between methods. We demonstrate the utility of this framework by performing 12 different methods in parallel to measure the same underlying reference system for cellular response. We compare method performance using quantitative evaluations of bias and resolvability. We attribute differences in method performance to steps along the measurement process such as sample preparation, signal detection, and choice of measurand. Finally, we demonstrate how this framework can be used to benchmark different methods for single-transcript detection. The framework we present here provides a practical way to compare performance of any methods.

Single-cell measurement of plasmid copy number and promoter activity.

Accurate measurements of promoter activities are crucial for predictably building genetic systems. Here we report a method to simultaneously count plasmid DNA, RNA transcripts, and protein expression in single living bacteria. From these data, the activity of a promoter in units of RNAP/s can be inferred. This work facilitates the reporting of promoters in absolute units, the variability in their activity across a population, and their quantitative toll on cellular resources, all of which provide critical insights for cellular engineering.

Measurements drive progress in directed evolution for precise engineering of biological systems.

Precise engineering of biological systems requires quantitative, high-throughput measurements, exemplified by progress in directed evolution. New approaches allow high-throughput measurements of phenotypes and their corresponding genotypes. When integrated into directed evolution, these quantitative approaches enable the precise engineering of biological function. At the same time, the increasingly routine availability of large, high-quality data sets supports the integration of machine learning with directed evolution. Together, these advances herald striking capabilities for engineering biology.

Cyberbiosecurity for Biopharmaceutical Products.

Cyberbiosecurity is an emerging discipline that addresses the unique vulnerabilities and threats that occur at the intersection of cyberspace and biotechnology. Advances in technology and manufacturing are increasing the relevance of cyberbiosecurity to the biopharmaceutical manufacturing community in the United States. Threats may be associated with the biopharmaceutical product itself or with the digital thread of manufacturing of biopharmaceuticals, including those that relate to supply chain and cyberphysical systems. Here, we offer an initial examination of these cyberbiosecurity threats as they stand today, as well as introductory steps toward paths for mitigation of cyberbiosecurity risk for a safer, more secure future.

Connecting nanoscale images of proteins with their genetic sequences.

We present a technique for reconstructing biomolecular structures from scanning force microscope data. The technique works by iteratively refining model molecules by comparison of simulated and experimental images. It can remove instrument artifacts to yield accurate dimensional measurements from tip-broadened data. The result of the reconstruction is a model that can be chosen to include the physically significant parameters for the system at hand. We demonstrate this by reconstructing scanning force microscope images of the cartilage proteoglycan aggrecan. By explicitly including the protein backbone in the model, we are able to associate measured three-dimensional structures with sites in the protein primary structure. The distribution of aggrecan core protein lengths that we measure suggests that 48% of aggrecan molecules found in vivo have been partially catabolized at either the E(1480)-(1481)G or E(1667)-(1668)G aggrecanase cleavage site.