Characterization of Wild type and mutant initiation/elongation complexes in Eschericia coli and Mycobacterium tuberculosis using Atomic Force Microscopy and Optical Tweezers """"""""This research will be done primarily in Peru at the Universidad Peruana Cayetano Heredia in collaboration with Daniel G. Guerra, as an extension of NIH Grant No. R0137 GM 032543, active from 2008 to 2013"""""""" Abstract During the last few years we have developed novel methods and approaches to study transcription by RNA polymerase (RNAP) of Escherichia coli (Eco). Here we propose to extend these studies to emphasize several aspects of transcription initiation and elongation as the main regulation steps of gene expression and to extend the research to a medically important species, Mycobacterium tuberculosis (Mtb) and its rifampicin-resistant mutants, a health problem of high priority in Peru. We will employ an experimental platform to study the complex functioning of both Eco and Mtb enzymes in their many steps. Initiation will be directly observed by AFM imaging to chafracterize: (1) DNA binding, (2) open complex formation at specific promoters (3) open complex stability, (4) kinetics of promoter escape, (5) kinetics of sigma factor release. By using alternative promoter sequences, we will characterize the different conformational determinants that govern each one of these steps. We expect that the promoter DNA sequences, the interaction of regulators such as ppGpp and RNAP mutations will determine different subsets of conformations in the initiation complex that will solve current questions on the mechanism of transcription control. In parallel, the elongation of RNA transcripts will be observed in real-time using optical tweezers. We will measure the instantaneous velocity, processivity, pause entry, pause exit and arrest probabilities of single elongation complexes. These highly informative experimental platforms will be used to study RIF resistant mutant enzymes. By determining which transcription step is affected in the mutant enzymes, we hope to gain insight into various aspects of the in vivo translation by RIF resistant mutants. This characterization will be followed by a bottom-to-top approach using techniques such as double-mutations, culture in selective media and micro-arrays. Also, we propose to develop a platform to test and determine the mechanism of inhibition of new compounds on RNAP. Given the current absence of crystallographic Eco and Mtb structural data, single-molecule manipulation and visualization should be a powerful approach to guide the improvement of inhibitors as lead compounds for the development of new drugs. Peru, just as many other low-income countries, has a very high prevalence of TB and MDR-TB, especially concentrated in its capital, Lima. The UPCH leads highly advanced in situ biomedical research in clinical, epidemiological and genetic aspects of TB. We believe that the long term aim of controlling and defeating TB will come from the concurrence of highly advanced basic research as proposed here, development of novel drugs, and responsible healthcare strategies for management of the TB challenge.

Public Health Relevance

Characterization of Wild type and mutant initiation/elongation complexes in Eschericia coli and Mycobacterium tuberculosis using Atomic Force Microscopy and Optical Tweezers Narrative Rifampicin is a very successful antibiotic for the treatment of tuberculosis and other bacterial infections that targets RNA polymerase (RNAP), the 5-subunit enzyme complex in charge of the regulated expression of all genes in bacteria. We will study the spatial relations and dynamics of RNA polymerases and their DNA substrates in Eschericia coli and Mycobacterium tuberculosis using single-molecule visualization and manipulation techniques to observe separately their various transcriptional intermediates. This approach constitutes a highly informative experimental platform that should make it possible to test new inhibitors of the enzymes and to guide their improvement as drug lead compounds. Moreover, since RNAP controls the expression of all bacterial genes, the study of naturally-occurring rifampicin-resistant mutants will provide insights on the development of resistant strains which could lead to future strategies to limit the emergence and spread of drug resistance.

National Institute of Health (NIH)
Fogarty International Center (FIC)
Small Research Grants (R03)
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International and Cooperative Projects - 1 Study Section (ICP1)
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Sina, Barbara J
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Universidad Peruana Cayetano Heredia
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LIMA -31
Doniselli, Nicola; Rodriguez-Aliaga, Piere; Amidani, Davide et al. (2015) New insights into the regulatory mechanisms of ppGpp and DksA on Escherichia coli RNA polymerase-promoter complex. Nucleic Acids Res 43:5249-62