In all cells, proteins are synthesized by ribosomes, megadalton RNA-protein machines that use aminoacyl-tRNA (aa-tRNA) substrates to translate messenger RNA (mRNA). In recent years, tremendous progress has been made in elucidating the structure of the ribosome in the absence and presence of substrates and various translation factors. Despite this wealth of structural information, several important questions about translation elongation and the role of rRNA in this process remain open.
Aim 1 of this proposal is to determine the role of 16S rRNA in decoding. A number of mutations in 16S rRNA that increase misreading in vivo will be characterized in vitro. The data obtained may reveal how signaling between the 30S A site and elongation factor EF-Tu is mediated.
Aim 2 of this proposal is to determine the molecular basis of ribosomal "unlocking," which limits the rate of translocation. Nine inter-subunit bridge mutations and four 30S neck mutations will be thoroughly characterized with respect to factor-independent and factor-dependent translocation in vitro. The data obtained will identify those ribosome- ribosome contacts contributing to the energy barrier for translocation and may help uncover the molecular basis of unlocking. Ribosomes are a main target of antibiotics, and defects in translation are associated with a growing number of inherited human diseases and cancers. Insight gained by this project may lead to the development of novel antimicrobial drugs and/or treatments for one or more hereditary diseases.

Public Health Relevance

One of the largest challenges facing modern medicine is the emergence of antibiotic resistance, and many classes of medically useful antibiotics target the ribosome. Defects in protein synthesis have been attributed to many inherited human diseases, and a growing body of evidence suggests that alteration in ribosome biogenesis and/or activity plays an important role in the development of several cancers. Insight gained from this project may (1) aid efforts to develop novel antibiotics and/or therapy regimes to combat pathogens with multiple-drug resistance and (2) contribute to the treatment and/or prevention of one or more hereditary diseases and/or cancers.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
3R01GM072528-09S1
Application #
8850685
Study Section
Molecular Genetics A Study Section (MGA)
Program Officer
Bender, Michael T
Project Start
2005-04-01
Project End
2015-04-30
Budget Start
2013-04-01
Budget End
2015-04-30
Support Year
9
Fiscal Year
2014
Total Cost
$96,739
Indirect Cost
$29,864
Name
Ohio State University
Department
Microbiology/Immun/Virology
Type
Schools of Arts and Sciences
DUNS #
832127323
City
Columbus
State
OH
Country
United States
Zip Code
43210
McClory, Sean P; Devaraj, Aishwarya; Fredrick, Kurt (2014) Distinct functional classes of ram mutations in 16S rRNA. RNA 20:496-504
Xia, Xin; Piao, Xijun; Fredrick, Kurt et al. (2014) Bifacial PNA complexation inhibits enzymatic access to DNA and RNA. Chembiochem 15:31-6
Fredrick, Kurt; Ibba, Michael (2014) The ABCs of the ribosome. Nat Struct Mol Biol 21:115-6
Liu, Qi; Fredrick, Kurt (2013) Contribution of intersubunit bridges to the energy barrier of ribosomal translocation. Nucleic Acids Res 41:565-74
Fagan, Crystal E; Dunkle, Jack A; Maehigashi, Tatsuya et al. (2013) Reorganization of an intersubunit bridge induced by disparate 16S ribosomal ambiguity mutations mimics an EF-Tu-bound state. Proc Natl Acad Sci U S A 110:9716-21
Shoji, Shinichiro; Janssen, Brian D; Hayes, Christopher S et al. (2010) Translation factor LepA contributes to tellurite resistance in Escherichia coli but plays no apparent role in the fidelity of protein synthesis. Biochimie 92:157-63
Devaraj, Aishwarya; Fredrick, Kurt (2010) Short spacing between the Shine-Dalgarno sequence and P codon destabilizes codon-anticodon pairing in the P site to promote +1 programmed frameshifting. Mol Microbiol 78:1500-9
Fredrick, Kurt; Ibba, Michael (2010) How the sequence of a gene can tune its translation. Cell 141:227-9
McClory, Sean P; Leisring, Joshua M; Qin, Daoming et al. (2010) Missense suppressor mutations in 16S rRNA reveal the importance of helices h8 and h14 in aminoacyl-tRNA selection. RNA 16:1925-34
Shoji, Shinichiro; Walker, Sarah E; Fredrick, Kurt (2009) Ribosomal translocation: one step closer to the molecular mechanism. ACS Chem Biol 4:93-107

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