The long-term objective of this research project is to understand how cells identify and recycle inactive ribosomes during protein synthesis. Not all ribosomes that initiate translation are able to complete synthesis of full-length proteins. In many instances, incomplete protein synthesis is due to "non-stop" mRNAs, which are truncated transcripts lacking in-frame stop codons. In bacteria, ribosomes trapped at the 34-ends of non-stop mRNA are "rescued" by the tmRNA (for transfer-messenger RNA) quality control system. Although tmRNA is ubiquitous throughout the eubacteria, it can be deleted from Escherichia coli cells due to the presence of a parallel tmRNA-independent ribosome rescue pathway. However, both pathways cannot be disrupted in E. coli, demonstrating that ribosome rescue is an essential function for these and probably all cells. This proposal focuses on three fundamental aspects of translational quality control that revolve around ribosome rescue. First, we seek to determine the functional relationship between A-site mRNA cleavage and ribosome rescue. A- site cleavage is a novel RNase activity that truncates mRNA in the A-site codon of stalled ribosomes, thereby producing non-stop mRNA. Biochemical and molecular genetic approaches will be used to identify the A-site nuclease. Identification of the A-site nuclease is necessary to determine whether the activity plays a functional role in either tmRNA-mediated or tmRNA-independent ribosome rescue. Second, we will characterize the recently identified tmRNA-independent rescue pathway, which appears to be mediated by YhdL. The biochemical function and regulation of YhdL will be investigated using in vitro and in vivo approaches. The spectrum of YhdL ribosome rescue activity will be identified, and its role in nascent chain release determined. Finally, the role of DnaK in tmRNA-mediated ribosome rescue will be mechanistically defined. DnaK activity will be ablated using molecular genetic and pharmacological approaches and the effects on tmRNA tagging assessed. A defined in vitro translation system will be used to determine whether DnaK exerts its effects by virtue of its co-translational chaperone activity.

Public Health Relevance

The protein synthesis machinery is a common target for antimicrobial drug therapy. This research seeks to uncover the molecular mechanisms underlying 'ribosome rescue', which is a general quality control response to defective protein synthesis in bacteria. Because ribosome rescue function is essential for bacterial survival, these pathways are potential targets for new antibiotics.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM078634-07
Application #
8324585
Study Section
Molecular Genetics A Study Section (MGA)
Program Officer
Bender, Michael T
Project Start
2006-07-01
Project End
2015-06-30
Budget Start
2012-07-01
Budget End
2013-06-30
Support Year
7
Fiscal Year
2012
Total Cost
$269,153
Indirect Cost
$79,153
Name
University of California Santa Barbara
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
094878394
City
Santa Barbara
State
CA
Country
United States
Zip Code
93106
Koskiniemi, Sanna; Garza-Sánchez, Fernando; Sandegren, Linus et al. (2014) Selection of orphan Rhs toxin expression in evolved Salmonella enterica serovar Typhimurium. PLoS Genet 10:e1004255
Whitney, John C; Beck, Christina M; Goo, Young Ah et al. (2014) Genetically distinct pathways guide effector export through the type VI secretion system. Mol Microbiol 92:529-42
Hayes, Christopher S; Koskiniemi, Sanna; Ruhe, Zachary C et al. (2014) Mechanisms and biological roles of contact-dependent growth inhibition systems. Cold Spring Harb Perspect Med 4:
Beck, Christina M; Diner, Elie J; Kim, Jeff J et al. (2014) The F pilus mediates a novel pathway of CDI toxin import. Mol Microbiol 93:276-90
Ruhe, Zachary C; Low, David A; Hayes, Christopher S (2013) Bacterial contact-dependent growth inhibition. Trends Microbiol 21:230-7
Koskiniemi, Sanna; Lamoureux, James G; Nikolakakis, Kiel C et al. (2013) Rhs proteins from diverse bacteria mediate intercellular competition. Proc Natl Acad Sci U S A 110:7032-7
Ruhe, Zachary C; Wallace, Adam B; Low, David A et al. (2013) Receptor polymorphism restricts contact-dependent growth inhibition to members of the same species. MBio 4:
Webb, Julia S; Nikolakakis, Kiel C; Willett, Julia L E et al. (2013) Delivery of CdiA nuclease toxins into target cells during contact-dependent growth inhibition. PLoS One 8:e57609
Janssen, Brian D; Garza-Sanchez, Fernando; Hayes, Christopher S (2013) A-site mRNA cleavage is not required for tmRNA-mediated ssrA-peptide tagging. PLoS One 8:e81319
Janssen, Brian D; Hayes, Christopher S (2012) The tmRNA ribosome-rescue system. Adv Protein Chem Struct Biol 86:151-91

Showing the most recent 10 out of 28 publications