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 i s 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 i s 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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM094157-03
Application #
8325081
Study Section
Macromolecular Structure and Function B Study Section (MSFB)
Program Officer
Flicker, Paula F
Project Start
2010-09-15
Project End
2015-08-31
Budget Start
2012-09-01
Budget End
2013-08-31
Support Year
3
Fiscal Year
2012
Total Cost
$304,722
Indirect Cost
$116,622
Name
Brown University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
001785542
City
Providence
State
RI
Country
United States
Zip Code
02912
Demirci, Hasan; Murphy 4th, Frank V; Murphy, Eileen L et al. (2014) Structural analysis of base substitutions in Thermus thermophilus 16S rRNA conferring streptomycin resistance. Antimicrob Agents Chemother 58:4308-17
Gregory, Steven T; Connetti, Jacqueline L; Carr, Jennifer F et al. (2014) Phenotypic interactions among mutations in a Thermus thermophilus 16S rRNA gene detected with genetic selections and experimental evolution. J Bacteriol 196:3776-83
Demirci, Hasan; Sierra, Raymond G; Laksmono, Hartawan et al. (2013) Serial femtosecond X-ray diffraction of 30S ribosomal subunit microcrystals in liquid suspension at ambient temperature using an X-ray free-electron laser. Acta Crystallogr Sect F Struct Biol Cryst Commun 69:1066-9
Cantara, William A; Murphy 4th, Frank V; Demirci, Hasan et al. (2013) Expanded use of sense codons is regulated by modified cytidines in tRNA. Proc Natl Acad Sci U S A 110:10964-9
Demirci, Hasan; Murphy 4th, Frank; Murphy, Eileen et al. (2013) A structural basis for streptomycin-induced misreading of the genetic code. Nat Commun 4:1355