Transcriptional regulation through DNA looping has been the main focus of our research for some time. Loop formation and breakdown can be seen as an efficient molecular switch and a feedback mechanism with which to sample the environment and respond via different genetic expression pathways.
Protein-induced DNA looping must operate amidst DNA bending, wrapping and compaction, and in the cell occurs on DNA segments that might be supercoiled and/or under tension. We, therefore, commonly use single molecule assays such as the tethered particle motion technique (TPM), and magnetic tweezers, which allow us to monitor and manipulate all these physical parameters along with the biochemistry that affects nucleoprotein complex formation.
Complementary AFM studies, in collaboration with Dr. Dunlap in the Cell Biology department of Emory Medical School, are carried out to elucidate the structure of protein/DNA complexes
Main Reserach projects under investigation:
Characterization of the bacteriophage Lambda regulattory loop. After the infection of Escherichia coli bacteria by lambda, the phage can follow either one of two different pathways: 1) lysogeny, when the viral DNA integrates into the host bacterial DNA and duplicates when the bacterium divides and 2) lysis, when the virus uses the bacterial molecular machinery to produce many viral copies before lysing the host cell. Once the virus enters the lysogenic state, it can be induced to switch to lytic growth through a process called prophage induction. However, lysogeny is extremely stable: recent data indicate that fewer than one in 107 -108 lysogenic cells spontaneously lyse per generation. Despite the extreme stability of the lysogen, prophage induction takes place with essentially 100% efficiency.
Lysogeny is stably maintained in the absence of an inducing signal (e.g. UV irradiation), but the system is poised to efficiently change upon receiving the signal to the alternative pattern of gene expression. In the prophage state, all the phage promoters, except the one that transcribes the CI gene, are turned off. By contrast, during lytic growth, most other phage genes are expressed. The CI repressor protein has a key role in the &lambda genetic switch. CI is both an activator and a repressor of transcription and is required for the maintenance of the lysogenic state. During lysogeny, dimers of CI bind to specific sites within the OL and OR control regions, located about 2.3 kbp apart on the phage genome (figure 1). Each control region contains three operators, OL1, OL2, OL3 and OR1, OR2, OR3. The cooperative binding of CI to OR1 and OR2 represses transcription from the PR promoter. Similarly, the binding of CI to OL1 and OL2 represses transcription from the PL promoter. By doing so, tetramers of CI inhibit the transcription of the phage’s lytic genes from PR and PL and, simultaneously, activate the transcription of the cI gene from the promoter PRM . It has recently been discovered that CI is also able to negatively regulate its own synthesis when present at high concentrations. This was explained by a model, proposed by Dodd and collaborators, suggesting that tetramers of CI bound to OL1, OL2 and OR1, OR2 interact to form an octamer and the intervening DNA sequence forms a loop. This higher–order DNA structure juxtaposes OL3 and OR3 so that a CI dimer bound at OL3 can stabilize a CI dimer bound at OR3, resulting in the repression of PRM. This model of negative autoregulation would limit CI concentration in the lysogenic state and permit an efficient switching to the lytic stage. We obtained the first direct observation of CI-induced DNA loop formation and breakdown (C. Zurla et al. JPCM, 2006), and we are currently investigating the molecular mechanism of such reaction. This work is in collaboration with the lab of Sankar Adhya at NCI, NIH.
Characterization of the equilibrium between DNA wrapping and looping in bacteriophage 186 and its role in the coliphage epigenetic switch. The current model for DNA binding by the 186 repressor protein (186 CI) is that this protein forms a planar ring of seven dimers - a 'wheel'- with a diameter of ~ 15 nm. the seven DNA-binding modules are arranged on the outside rim, allowing up tp ~ 130 bp(~ 12helical turns or ~ 41 nm) of contiguous DNA to wrap around the wheel by binding up to six of the CI dimers. A CI wheel can also be occupied by non-contiguous DNA, causing the formation of DNA loops of at least 300 bp (102 nm). Transcriptional regulation by CI is achieved by competition between various alternative wrapped and looped DNA structure. If this model is correct, then the 186 CI-DNA interaction will provide a tractable experimental system for examination of the parameters that determined the formation and behavior of wrapped and looped DNA structure in vitro and in vivo . The goal is to test the model by examining: (1) CI-DNA wrapping, and (2) CI-DNA looping by using three different in vitro single-molecule detection and manipulation assays. The comparison with in vivo results obtained by collaborators will allow to directly relate the structure and the molecular mechanism determined at the single-molecule level with the in vivo physiology.
This work is in collaboration with Drs. Keith Shearwin and Ian Dodd at the University of Adelaide in Australia and with Dr. David Dunlap in Cell Biology.
DNA flexibility. In collaboration with Dr. David Dunlap in the Cell Biology Department, we study how variations in the bending rigidity of DNA may affect motor proteins.
Transscriptional regulation by the Male Specific Lethal (MSL) complex of Drosophila. In collaboration with Dr. Lucchesi in Biology Department, we are also studying the molecular properties of the Male Specific Lethal (MSL) complex of Dorsophila. This complex enhances the level of transcription of numerous genes on the X chromosome of Drosophila males and ensures dosage compensation in males and females. Dosage compensation is a fundamental problem of transcriptional regulation, epigenetics and developmental biology. The MSL complex contains an ATP-dependent RNA/DNA helicase (MLE). In Drosophila males, the complex assembles on the X chromosomes at specific sites and then spreads to munerous additional sites. From these sites, complexes target activated genes in order to increase their level of expression. Our laboratory uses single-molecule assays and bulk biophysical techniques to characterize the paramenters that are responsible for the translocation of the MSL complex along DNA.
Instrumentation development:
This is an active area of investigation in the laboratory, since questions often require new instrumentation. For example, we recently developed a novel single molecule microscope that allow simultaneous observation of DNA conformations and protein activity to determine how certain motor enzymes are affected by protein-induced conformational changes in DNA.
Theory:
The lab is engaged in an ongoing effort to optimize the procedure used to derive lifetimes from the experimental data. We are developing and comparing methods based on auto-correlation functions, threshold determinations, etc. Furthermore the systems investigated, and the kinetic and thermodynamic information accessed through single-molecule experimentation provide fertile ground for mechanistic modeling. We formulate ad hoc computational and analytical models independently and in collaboration with theoretical physicists in order to validate, explain and interpret the experimental data.
Characterization of the bacteriophage Lambda regulattory loop. After the infection of Escherichia coli bacteria by lambda, the phage can follow either one of two different pathways: 1) lysogeny, when the viral DNA integrates into the host bacterial DNA and duplicates when the bacterium divides and 2) lysis, when the virus uses the bacterial molecular machinery to produce many viral copies before lysing the host cell. Once the virus enters the lysogenic state, it can be induced to switch to lytic growth through a process called prophage induction. However, lysogeny is extremely stable: recent data indicate that fewer than one in 107 -108 lysogenic cells spontaneously lyse per generation. Despite the extreme stability of the lysogen, prophage induction takes place with essentially 100% efficiency.
Lysogeny is stably maintained in the absence of an inducing signal (e.g. UV irradiation), but the system is poised to efficiently change upon receiving the signal to the alternative pattern of gene expression. In the prophage state, all the phage promoters, except the one that transcribes the CI gene, are turned off. By contrast, during lytic growth, most other phage genes are expressed. The CI repressor protein has a key role in the &lambda genetic switch. CI is both an activator and a repressor of transcription and is required for the maintenance of the lysogenic state. During lysogeny, dimers of CI bind to specific sites within the OL and OR control regions, located about 2.3 kbp apart on the phage genome (figure 1). Each control region contains three operators, OL1, OL2, OL3 and OR1, OR2, OR3. The cooperative binding of CI to OR1 and OR2 represses transcription from the PR promoter. Similarly, the binding of CI to OL1 and OL2 represses transcription from the PL promoter. By doing so, tetramers of CI inhibit the transcription of the phage’s lytic genes from PR and PL and, simultaneously, activate the transcription of the cI gene from the promoter PRM . It has recently been discovered that CI is also able to negatively regulate its own synthesis when present at high concentrations. This was explained by a model, proposed by Dodd and collaborators, suggesting that tetramers of CI bound to OL1, OL2 and OR1, OR2 interact to form an octamer and the intervening DNA sequence forms a loop. This higher–order DNA structure juxtaposes OL3 and OR3 so that a CI dimer bound at OL3 can stabilize a CI dimer bound at OR3, resulting in the repression of PRM. This model of negative autoregulation would limit CI concentration in the lysogenic state and permit an efficient switching to the lytic stage. We obtained the first direct observation of CI-induced DNA loop formation and breakdown (C. Zurla et al. JPCM, 2006), and we are currently investigating the molecular mechanism of such reaction. This work is in collaboration with the lab of Sankar Adhya at NCI, NIH.
Characterization of the equilibrium between DNA wrapping and looping in bacteriophage 186 and its role in the coliphage epigenetic switch. The current model for DNA binding by the 186 repressor protein (186 CI) is that this protein forms a planar ring of seven dimers - a 'wheel'- with a diameter of ~ 15 nm. the seven DNA-binding modules are arranged on the outside rim, allowing up tp ~ 130 bp(~ 12helical turns or ~ 41 nm) of contiguous DNA to wrap around the wheel by binding up to six of the CI dimers. A CI wheel can also be occupied by non-contiguous DNA, causing the formation of DNA loops of at least 300 bp (102 nm). Transcriptional regulation by CI is achieved by competition between various alternative wrapped and looped DNA structure. If this model is correct, then the 186 CI-DNA interaction will provide a tractable experimental system for examination of the parameters that determined the formation and behavior of wrapped and looped DNA structure in vitro and in vivo . The goal is to test the model by examining: (1) CI-DNA wrapping, and (2) CI-DNA looping by using three different in vitro single-molecule detection and manipulation assays. The comparison with in vivo results obtained by collaborators will allow to directly relate the structure and the molecular mechanism determined at the single-molecule level with the in vivo physiology.
This work is in collaboration with Drs. Keith Shearwin and Ian Dodd at the University of Adelaide in Australia and with Dr. David Dunlap in Cell Biology.
DNA flexibility. In collaboration with Dr. David Dunlap in the Cell Biology Department, we study how variations in the bending rigidity of DNA may affect motor proteins.
Transscriptional regulation by the Male Specific Lethal (MSL) complex of Drosophila. In collaboration with Dr. Lucchesi in Biology Department, we are also studying the molecular properties of the Male Specific Lethal (MSL) complex of Dorsophila. This complex enhances the level of transcription of numerous genes on the X chromosome of Drosophila males and ensures dosage compensation in males and females. Dosage compensation is a fundamental problem of transcriptional regulation, epigenetics and developmental biology. The MSL complex contains an ATP-dependent RNA/DNA helicase (MLE). In Drosophila males, the complex assembles on the X chromosomes at specific sites and then spreads to munerous additional sites. From these sites, complexes target activated genes in order to increase their level of expression. Our laboratory uses single-molecule assays and bulk biophysical techniques to characterize the paramenters that are responsible for the translocation of the MSL complex along DNA.
Instrumentation development:
This is an active area of investigation in the laboratory, since questions often require new instrumentation. For example, we recently developed a novel single molecule microscope that allow simultaneous observation of DNA conformations and protein activity to determine how certain motor enzymes are affected by protein-induced conformational changes in DNA.
Theory:
The lab is engaged in an ongoing effort to optimize the procedure used to derive lifetimes from the experimental data. We are developing and comparing methods based on auto-correlation functions, threshold determinations, etc. Furthermore the systems investigated, and the kinetic and thermodynamic information accessed through single-molecule experimentation provide fertile ground for mechanistic modeling. We formulate ad hoc computational and analytical models independently and in collaboration with theoretical physicists in order to validate, explain and interpret the experimental data.
