2007 — 2009 |
Jogl, Gerwald |
P20Activity Code Description: To support planning for new programs, expansion or modification of existing resources, and feasibility studies to explore various approaches to the development of interdisciplinary programs that offer potential solutions to problems of special significance to the mission of the NIH. These exploratory studies may lead to specialized or comprehensive centers. |
Structural Studies of Phosphoinositide Related Protein Kinases |
1 |
2010 |
Jogl, Gerwald |
P41Activity Code Description: Undocumented code - click on the grant title for more information. |
Post-Transcriptional Rrna Modification For the 30s Ribosomal Subunit
This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Post-transcriptional and post-translational modification of ribosomal RNA and proteins is common in most or-ganisms, but the function of many of these modifications is not known. In bacteria, loss of some modifications leads to increased or decreased resistance to antibiotic drugs. We investigate the significance of these modi-fications for ribosome structure and function. This project is carried out in collaboration with Albert Dahlberg at Brown University, Venki Ramakrishnan at the MRC, UK, and Frank Murphy at the APS. We have now produced crystals for 30S ribosomal subunits from T. thermophilus with the gene for the rRNA methyltrans-ferase KsgA deleted. The KsgA dimethyltransferase modifies residues A1518 and A1519 of the 16S rRNA. This modification appears to be conserved in all organisms and confers sensitivity to the antibiotic kasu-gamycin. Structural information for the unmodified 30S subunits will lead to a better understanding of the function of these modifications. In a second approach, we are studying the impact of mutations on ribosome structure. We have produced 30S subunit crystals for two mutant forms: the streptomycin-dependent mutant G524U and the reverting double mutant G524U / A10G. We plan to determine structures of the mutant 30S ribosomal subunits in the apo-form and in complex with streptomycin to understand the rearrangements that lead to the altered binding of the antibiotic in the mutant forms.
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0.966 |
2010 — 2021 |
Gregory, Steven (co-PI) [⬀] Gregory, Steven (co-PI) [⬀] Jogl, Gerwald |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Structural Robustness of Ribosome Functional Centers
DESCRIPTION (provided by applicant): The ribosome is the universal site of protein synthesis, containing some of the most highly conserved of all biological sequences. Nevertheless, the ribosome is robust to mutation, capable of functioning when challenged with base or amino acid substitutions in its highly conserved functional centers. As major targets of antibiotics, these functional centers are the sites of numerous antibiotic-resistance mutations. While it has been well established that antibiotic-Resistance mutations carry a substantial fitness cost, the structural basis for this burden is only now within the scope of our technical ability to investigate. In this proposal, we describe a synthetic approach using genetics, chemical probing and X-ray crystallography of ribosomes from the thermophilic bacterium Thermus thermophilus to address the structural robustness of ribosome active sites and its relationship to biological fitness. Our development of T. thermophilus ribosome genetics has enabled us to identify or construct antibiotic-resistant mutants at will. We also now have the technical ability to crystallize wild-type and mutant 30S ribosomal subunits and 70S ribosomes and to determine their three-dimensional structures by X-ray diffraction. Together with the development of novel chemical probing techniques to investigate RNA conformational dynamics, these abilities have placed us in a unique position to address three specific issues. The first aim of our proposal is to use streptomycin-resistance mutations as a paradigm for examining the mutational robustness of a conserved ribosome functional center that participates in global conformational changes of the 30S subunit. Our second aim is to use tuberactinomycin-resistance to examine the effects of mutations on the structure and function of an intersubunit bridge that is critical for large-scale rotational motions of the entire 70S ribosome. The third aim is to use deleterious antibiotic-resistance mutations in the peptidyltransferase active site to evolve compensatory mutations that restore fitness, and to examine their structural effects using X-ray crystallography. The goal of this aim is to detect as yet unrecognized long-range functional relationships throughout the ribosome. We will also use the peptidyltransferase active site to examine the limits of robustness of ribosome functional centers to mutation. In addition to providing a more complete mechanistic understanding of antibiotic resistance at an unprecedented level of resolution, these efforts are directed towards establishing fundamental principles of ribosome structural organization and evolution. PUBLIC HEALTH RELEVANCE: The goal of this project is to study the impact of antibiotic-resistance mutations upon ribosome structure and function in order to gain a better understanding of the molecular mechanism of resistance. Results from these studies will provide valuable information for the rational development of new ribosome-targeting antibiotic compounds to combat pathogens that are resistant to currently available drugs.
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1 |
2011 |
Jogl, Gerwald |
P41Activity Code Description: Undocumented code - click on the grant title for more information. |
Structural Robustness of the Ribosome
This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The ribosome is the universal site of protein synthesis, containing some of the most highly conserved of all biological sequences. Nevertheless, the ribosome is robust to mutation, capable of functioning when chal-lenged with base or amino acid substitutions in its highly conserved functional centers. As major targets of antibiotics, these functional centers are the sites of numerous antibiotic-resistance mutations. While it has been well established that antibiotic-resistance mutations carry a substantial fitness cost, the structural basis for this burden is only now within the scope of our technical ability to investigate. Here, we will use X-ray crystallography of ribosomes from the thermophilic bacterium Thermus thermophilus to address the structural robustness of ribosome active sites and its relationship to biological fitness. In collaboration with Albert Dahl-berg and Steven Gregory at Brown University, we will investigate streptomycin-resistance mutations to ex-amine the mutational robustness of a conserved ribosome functional center that participates in global con-formational changes of the 30S subunit. In collaboration with Frank Murphy, we have already collected dif-fraction data sets for 30S subunit crystals carrying streptomycin-resistance mutations and the streptomycin-dependence mutation G524U. Overall, we have crystals available for 30S subunits carrying several antibi-otic-resistance mutations and we are in the process of preparing a large number of additional ribosome sam-ples. In addition to providing a more complete mechanistic understanding of antibiotic resistance at an un-precedented level of resolution, these efforts are directed towards establishing fundamental principles of ri-bosome structural organization and evolution.
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0.966 |