Regulation of Mycobacterial Transcription Initiation

Cellular life relies on gene expression, in which DNA is first transcribed into RNA, which is then translated into protein. Transcription is performed by the protein RNA polymerase (RNAP), which interacts with DNA in three sequential events known as initiation, elongation and termination. Mycobacterium tuberculosis (Mtb) represents a global burden to public health, and the transcription factors CarD and RbpA are both essential to Mtb. I have studied the effect of CarD and RbpA on transcription initiation in vitro at the Mtb rrnAP3 promoter. I have shown that CarD stabilizes unwinding of promoter DNA by RNAP using a two-tiered kinetic mechanism. I have also shown that RbpA stabilizes mycobacterial open complexes using a mechanism distinct from that of CarD. Furthermore, RbpA and CarD cooperatively stabilize mycobacterial open complexes, leading to increased transcription. Taken together, these findings lay the groundwork for a mechanistic understanding of gene regulation by essential transcription factors in a bacterium that kills millions of people each year

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 RP(o) 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 RP(o) 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 RP(o) on rrnAP3, suggesting an important difference between the polymerases themselves and highlighting how transcriptional machinery can vary across bacterial genera

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

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 (RP(o)) 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 RP(o). 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

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

Summary: 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)

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

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