The long-term goal of this grant proposal is to understand at an atomic level how the ribosome functions during protein synthesis. We will take a multidisciplinary approach utilizing genetic, biochemical and structural methods to study the bacterial ribosome.
Our first aim i s to examine ribosome structural dynamics during decoding of mRNA and its inhibition by antibiotics. We will use X-ray crystallography to obtain high-resolution structures of mutant Thermus thermophilus 30S subunits that reveal new conformational states. We will examine the effects of the mutations on the accuracy and kinetics of decoding.
Our second aim i s to probe a pathway by which codon recognition is signaled via ribosomal protein S12 and aminoacyl-tRNA to EF-Tu to activate GTP hydrolysis, a key step in the tRNA selection process. These experiments are designed to provide fundamental insights into ribosome structure and function, including new conformational states of the ribosome, to aid in understanding the decoding process. The ability to solve structures of ribosomes with antibiotic-resistance mutations will aid in understanding the mechanisms of antibiotic action and resistance, and has particular clinical relevance for the rational design of drugs to fight resistant microbial pathogens.

Public Health Relevance

PROJECT NARRATIVE: The goal of this project is to further our understanding of the molecular mechanisms of protein synthesis at the atomic level, using the methods of structural biology, genetics and biochemistry. Protein synthesis in bacteria is the target of over half of all antibiotics, and modifications and mutations in components involved of this process can lead to antibiotic resistance, one of the most pressing medical problems of our time. Deciphering the molecular basis for antibiotic resistance will provide important information needed for the rational design of novel antimicrobial agents effective against antibiotic resistant microorganisms. A more comprehensive examination of the structural basis for protein synthesis will also provide important insights into one of the most fundamental processes found in all forms of life.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM019756-41
Application #
8450936
Study Section
Molecular Genetics A Study Section (MGA)
Program Officer
Bender, Michael T
Project Start
1975-09-01
Project End
2015-03-31
Budget Start
2013-04-01
Budget End
2015-03-31
Support Year
41
Fiscal Year
2013
Total Cost
$634,268
Indirect Cost
$242,745
Name
Brown University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
001785542
City
Providence
State
RI
Country
United States
Zip Code
02912
Carr, Jennifer F; Gregory, Steven T; Dahlberg, Albert E (2015) Transposon mutagenesis of the extremely thermophilic bacterium Thermus thermophilus HB27. Extremophiles 19:221-8
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
Demirci, Hasan; Wang, Leyi; Murphy 4th, Frank V et al. (2013) The central role of protein S12 in organizing the structure of the decoding site of the ribosome. RNA 19:1791-801
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
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
Monshupanee, Tanakarn; Johansen, Shanna K; Dahlberg, Albert E et al. (2012) Capreomycin susceptibility is increased by TlyA-directed 2'-O-methylation on both ribosomal subunits. Mol Microbiol 85:1194-203
Demirci, Hasan; Murphy 4th, Frank; Belardinelli, Riccardo et al. (2010) Modification of 16S ribosomal RNA by the KsgA methyltransferase restructures the 30S subunit to optimize ribosome function. RNA 16:2319-24
Demirci, Hasan; Belardinelli, Riccardo; Seri, Emilia et al. (2009) Structural rearrangements in the active site of the Thermus thermophilus 16S rRNA methyltransferase KsgA in a binary complex with 5'-methylthioadenosine. J Mol Biol 388:271-82
Demirci, Hasan; Gregory, Steven T; Dahlberg, Albert E et al. (2008) Multiple-site trimethylation of ribosomal protein L11 by the PrmA methyltransferase. Structure 16:1059-66
Kapralou, Stavroula; Fabbretti, Attilio; Garulli, Chiara et al. (2008) Translation initiation factor IF1 of Bacillus stearothermophilus and Thermus thermophilus substitute for Escherichia coli IF1 in vivo and in vitro without a direct IF1-IF2 interaction. Mol Microbiol 70:1368-77

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