Browsing by Author "Schumacher, Maria A"

Replicative errors contribute to the genetic diversity needed for evolution but in high frequency can lead to genomic instability. The Watson-Crick base pairs that ensure the fidelity of the genome generation after generation adopt a Watson-Crick geometry that is defined by their shape complementarity as a function of their hydrogen bonding patterns and their inter-nucleotide distance. The ability of a base pair to adopt the Watson-Crick geometry can determine its ability to be incorporated by the polymerase. Conversely, mismatches which can adopt a Watson-Crick-like geometry can also be incorporated by the polymerases. These Watson-Crick-like mismatches have been hypothesized to be lowly-populated and short-lived alternative conformations, which are difficult to characterize and visualize with most biophysical techniques. In this thesis, using NMR spectroscopy and kinetic simulations, we determined the ground state conformation of the A•G mismatch, characterized the formation of Watson-Crick-like Hoogsteen excited state conformations in DNA duplexes and investigated their role in DNA replication errors. By including a DNA-dynamics-driven step into a minimal kinetic mechanism for correct incorporation after the initial nucleotide binding step, we were able to accurately recapitulate the rate with which A•dGTP mismatches are misincorporated for polymerase β, across different pH conditions, and for the most damaged form of the mismatch involving 8-oxoguanine. We further find that the introduction of 8-oxoguanine to A•G does not stabilize the mismatch to only one conformation but rather redistributes the dynamic ensemble of the mismatch to favor the mutagenic conformation as the ground state. Through direct and indirect targeting of the 8-oxoguanine and adenine, respectively, the lesion not only affects the mismatch itself but also rescues long-range dynamics of the DNA duplex that was previously repressed by the unmodified mismatch. We also characterize the sequence-dependent dynamics of the A•G mismatch and find that the nearest-neighbor base changes can significantly alter the dynamic ensemble. These sequence-dependent modulations to the conformational landscape can explain the sequence-dependent behavior of MutY repair enzyme.

Francisella tularensis is one of the most infectious bacteria known and is the etiologic agent of tularemia. Francisella virulence arises from a 33 kilobase pathogenicity island (FPI) that is regulated by the macrophage growth locus protein A (MglA), the stringent starvation protein A (SspA) and the pathogenicity island gene regulator (PigR). MglA•SspA•PigR interacts with the RNA polymerase (RNAP) to activate FPI transcription. The activity of MglA•SspA•PigR•RNAP is mediated by ppGpp, the alarmone of the stringent response. However, the molecular mechanisms involved in FPI transcriptional regulation are not well understood.

While most bacterial SspA proteins interact with the RNAP as a homodimer, F. tularensis SspA forms a heterodimer with MglA, which is unique to F. tularensis. To gain insight into MglA function, we performed structural and biochemical studies. The MglA structure revealed that it contains a fold similar to the SspA protein family, and unexpectedly formed a homodimer in the crystal. Chemical crosslinking and size exclusion chromatography (SEC) studies showed that, while it can self-associate in solution to form a dimer, MglA preferentially forms a heterodimer with SspA. As research with MglA highlighted that MglA•SspA is likely the only functional SspA protein in Francisella, X-ray crystallography was used to determine the structure of MglA•SspA to 2.65 Å. Analysis of the MglA•SspA structure revealed a vast hydrogen bond network at the interface that is significantly larger than the network at the MglA homodimerization interface. This difference could explain why MglA and SspA form a stable heterodimer and MglA (in the absence of SspA) forms a transient homodimer.

To gain insight into the molecular mechanism by which ppGpp mediates FPI transcription, differential radial capillary action of ligand assay (DRaCALA) was performed to determine which Francisella protein binds ppGpp. The DRaCALA experiments revealed that the MglA•SspA complex binds ppGpp specifically and with high affinity, while PigR, MglA and E. coli SspA do not. To characterize this interaction the structure of MglA•SspA was determined in complex with ppGpp (MglA•SspA•ppGpp) to 2.8 Å resolution, and revealed a single ppGpp molecule, which is also coordinated by Mg2+, bound per heterodimer. Analysis of structure-guided MglA•SspA ppGpp-binding mutants by DRaCALA confirmed the crystallographic binding site of ppGpp. Intriguingly, the binding of ppGpp does not produce a marked conformational change in MglA•SspA, but as the ppGpp-binding site overlaps with the previously ascribed PigR interaction surface it seems likely that ppGpp mediates the interaction between MglA•SspA and PigR.

The interaction between PigR and MglA•SspA is largely uncharacterized. Using fluorescence anisotropy we sought to determine which portion of PigR is responsible for interactions with MglA•SspA. Using fluoresceinated PigR peptides we determined that the final 22 residues of the PigR C-terminus interact with MglA•SspA. These results were corroborated using bridge-hybrid experiments by our collaborators. Further, as FA performed with and without ppGpp shows no difference in affinity between MglA•SspA and PigR, ppGpp must mediate this interaction through an as of yet undetermined mechanism, which is supported by very different millipolarization values indicating perhaps alternative binding modes of PigR.

All together, our results contribute significantly to our understanding of FPI transcriptional regulation.

DNA partition or segregation is the process that ensures the stable inheritance of genomic material. The majority of the bacterial plasmid and some chromosomal partition systems utilize ParA Walker-box-based partition systems. These systems require three components: a DNA centromere site, the ParA ATPase, and the ParB centromere binding protein. ParB binds to the centromere to form the partition complex, which then recruits the motor protein ParA. ParA mediates the partition of replicated DNA by a still poorly understood mechanism. Notably, recent data indicates that ParA Walker-box-based partition systems are employed not only by bacterial plasmids and chromosomes but also DNA elements in archaea. The work in this thesis focused on a homolog of the ParA protein from the first identified archaeal plasmid partition system, located on the plasmid pNOB8. pNOB8 plasmid is harbored in the thermophilic archaeaon, Sulfolobus solfataricus. The goals of this work were to structurally and biochemically characterize the ParA homolog to gain insights into its function.

Towards these goals, the structure of the ParA homolog was solved by X-ray crystallography in its apo and ADP bound states to resolutions of 2.45 Å and 2.73 Å, respectively. The overall structure was similar to bacterial ParA proteins. We next demonstrated that, similar to bacterial ParA proteins, this ParA homolog harbored ATP-dependent nonspecific DNA capabilities by using fluorescence polarization based DNA binding assays. By mutating the residues in the deviant Walker A motif, we were able to demonstrate the importance of ATP binding in its DNA binding function. Moreover, characterization of ATP and ADP binding were performed using ITC. Finally, we observed that ParA was able to form polymers in the presence of ATP, using negative stain electron microscopy. Our findings provide evidence that ParA Walker-box-based partition systems, which are the most common systems in bacteria, appear to also be found in archaea.

The typical flow of genetic information involves DNA being transcribed into RNA, which is then translated into protein. However, in some organisms this pathway requires an extra step where the RNA is post-transcriptionally modified before it can be translated. This process is referred to as RNA editing and can involve the substitution or the insertion and/or deletion of bases. Insertion/deletion RNA editing is rare, only occurring in the mitochondria of Physarum and trypanosomes, a group of parasitic protozoa. The RNA editing process in trypanosomes is called kinetoplastid RNA (kRNA) editing, after the kinetoplastid containing mitochondria, and creates translatable open reading frames by inserting and/or deleting uridine bases within the encoded mRNA sequence. The main proteins responsible for the kRNA editing process have been characterized and form a 20S editosome. Other accessory proteins have also been implicated in the editing process. Recently, the multiprotein mitochondrial RNA binding complex 1 (MRB1) has emerged as a key player in this process. One key component predicted to be involved in the MRB1 complex is MRB1590. In vivo experiments indicate a role for MRB1590 in editing mitochondrial mRNA transcripts, in particular the transcript encoding the ATP synthase subunit 6 (A6). The goals of this work were to structurally and biochemically characterize MRB1590 to characterize its role in the kRNA editing process, specifically in the editing of the A6 transcript.

The structure of MRB1590 was solved using X-ray crystallography under three different conditions. The overall structure shows the protein is dimeric and contains a central ABC-ATPase fold embedded between a novel N- and C-terminal domain, with a pore being created between the N-terminal domains. The structure of MRB1590 was solved in the presence of ADP and AMP-PNP, showing the nucleotide bound in a pocket located in the ABC-ATPase fold, similar to other ABC-ATPase proteins. The structure of MRB1590 solved in the presence of ADP and RNA revealed a distinct conformation compared to the other two structures in which it adopted an “open” state with a significantly expanded pore. These combined structures suggest that MRB1590 is in equilibrium between open and closed states with ADP binding favoring the open conformation allowing RNA to bind in the pore.

Fluorescence polarization (FP) experiments showed that MRB1590 binds with significantly enhanced affinity to a GC-rich RNA sequence from the A6 transcript compared to other RNA sequences that were tested. Because the MRB1590 structures predict the RNA binding pocket to be the pore created by N-terminal domains, basic residues present in this region were mutated and the effect on RNA binding was tested. The RNA binding of MRB1590 pore mutants was reduced, indicating that these residues are involved in RNA binding. Our findings support MRB1590 as a kRNA editing accessory protein and suggest it may act as an RNA chaperone that ensures the complete editing of the A6 transcript.

One of the most infectious bacteria in the world, the Gram-negative bacterium Francisella tularensis is the etiological agent of tularemia in humans. Francisella virulence stems from a gene cluster known as the Francisella pathogenicity island that encodes a type VI secretion system and enables it to escape from macrophages during an infection. Expression of the FPI is linked to the stringent response and is tightly controlled. Three transcription factors, macrophage growth locus protein A (MglA), stringent starvation protein A (SspA), and the pathogenicity island gene regulator (PigR), form a complex with RNA polymerase (RNAP) to activate transcription of the FPI. The alarmone of the stringent response, ppGpp, mediates the formation of the regulatory complex. MglA, which is unique to Francisella, forms a heterodimer with the conserved factor SspA, which functions as a homodimer in other Gram-negatives. Together, the MglA-SspA heterodimer associates with the σ70-containing RNAP using its closed face and binds ppGpp directly on the opposite face. With ppGpp bound, PigR can bind MglA-SspA with its C-terminal end while also making contacts to an upstream promoter DNA site known as the PigR response element (PRE) likely using its putative winged helix-turn-helix motif. Once the full complex is assembled, FPI transcription is activated through mechanisms that are not well understood. It is unclear how MglA-SspA, and PigR associate with RNAP and how these factors drive transcription at virulence promoters and, thus, enable Francisella to cause disease. To provide insight into the mechanisms of virulence activation by this unusual set of regulators, we employed single-particle cryo-EM, X-ray crystallography, and cellular analyses. First, we determined a cryo-EM structure of F. tularensis RNAPσ70 in complex with MglA-SspA and a virulence promoter which revealed the MglA-SspA binding mechanism. MglA-SspA does not contact the DNA, but instead interacts with two regions of the σ factor and makes an extensive hydrophobic interface with the RNAP core subunit β’. This effectively tethers the σ factor to the core enzyme to stabilize the RNAP holoenzyme and facilitate DNA binding. This structure adopted an open-complex; however, when we determined the structure with the same virulence promoter in the absence of MglA-SspA, the complex was unable to stably bind the DNA suggesting that MglA-SspA may itself regulate transcription at certain promoters by facilitating promoter binding and possibly open complex formation. We followed up on this with RNA sequencing studies that demonstrated MglA-SspA regulates many virulence and virulence-enhancing non-FPI genes independently of PigR. We next determined a cryo-EM reconstruction of E. coli RNAP in complex with an E. coli SspA homodimer and DNA which showed conservation of the σ-tethering mechanism. While we showed MglA-SspA alone can regulate a subset of virulence and virulence-enhancing genes, the activation of FPI genes requires PigR. We determined a crystal structure of (MglA-SspA)-ppGpp with a PigR C-terminal tail peptide. PigR makes direct contact to ppGpp through electrostatic interactions, which explains why ppGpp is needed for specific binding of PigR. Lastly, a cryo-EM structure of the full virulence complex consisting of Ftu RNAPσ70-(MglA-SspA)-ppGpp-PigR with an FPI promoter revealed an unexpected activation mechanism. This reconstruction revealed that PigR enhances transcription by recruiting the C-terminal domains of the RNAP core α subunits to previously unrecognized DNA UP elements that flank the PRE on both sides. All together, these findings have significantly advanced our understanding of virulence activation in F. tularensis and shed light on a general mechanism of transcription regulation by the SspA protein family.