Bacteria possess a unique system for rescuing aberrantly stalled ribosomes and marking for degradation the still linked, partially synthesized protein fragments. This quality control system, also known as transtranslation, is orchestrated by a remarkable RNA (SsrA RNA) that functions as a tRNA to detect and revive stalled ribosomes and as an mRNA to facilitate the addition of a short degradation tag to the C-terminus of nascent polypeptides. All known activities of SsrA require SmpB, a small protein that binds SsrA specifically and with high affinity to promote its association with stalled ribosomes. The molecular basis for the formation of the SmpB SsrA complex and the subsequent recognition of impaired ribosomes are not well understood. The objective of this research program is to use a combination of molecular genetics, protein biochemistry, bioinformatics, and structural approaches to elucidate the mechanism of the SmpB-SsrA quality control system. The emphasis is on the molecular characterization of how SmpB recognizes SsrA RNA and promotes the detection and rescue of stalled ribosomes. Principally, through these studies we wish to understand the biochemical and structural basis for the interactions of SmpB with SsrA RNA. Specifically we want to learn what amino acid residues are involved, what base-specific contacts are made, and what structural features contribute to the formation of the SmpB SsrA complex and its interaction with the ribosome. Furthermore, we wish to identify and characterize any additional cellular factors that might participate in this process. Specific complexes of RNA and protein perform many essential biological functions, including RNA processing, RNA turnover, RNA transport, RNA folding, as well as the translation of genetic information from mRNA into protein sequences. Principles that govern RNA-protein interactions are inadequately understood due in large part to a paucity of structural information on RNA-protein complexes. These principles are important for understanding RNA-protein machines, such as the ribosome, and RNA-protein structure and function in general. The relative simplicity of the SmpB-SsrA interaction, the stability of the complex, and recruitment of additional novel factors during trans-translation makes it an ideal system to study the basic principles underlying the assembly of RNA-protein complexes. Understanding of the RNA-protein assembly processes in this system are likely to provide new insights generalizable to the molecular mechanism of how RNA-binding proteins function. Moreover, because the SmpB SsrA quality control system exists only in prokaryotes and involves novel RNA and protein factors that are essential for the survival of most (if not all) pathogenic bacteria, a better understanding of this unique process might allow the design of highly specific new anti-bacterial agents.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM065319-05
Application #
7025084
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Program Officer
Rhoades, Marcus M
Project Start
2002-04-01
Project End
2008-07-31
Budget Start
2006-04-01
Budget End
2008-07-31
Support Year
5
Fiscal Year
2006
Total Cost
$252,777
Indirect Cost
Name
State University New York Stony Brook
Department
Biochemistry
Type
Schools of Medicine
DUNS #
804878247
City
Stony Brook
State
NY
Country
United States
Zip Code
11794
Friedlander, Lonia R; Puri, Neha; Schoonen, Martin A A et al. (2015) The effect of pyrite on Escherichia coli in water: proof-of-concept for the elimination of waterborne bacteria by reactive minerals. J Water Health 13:42-53
Venkataraman, Krithika; Guja, Kip E; Garcia-Diaz, Miguel et al. (2014) Non-stop mRNA decay: a special attribute of trans-translation mediated ribosome rescue. Front Microbiol 5:93
Venkataraman, Krithika; Zafar, Hina; Karzai, A Wali (2014) Distinct tmRNA sequence elements facilitate RNase R engagement on rescued ribosomes for selective nonstop mRNA decay. Nucleic Acids Res 42:11192-202
Camenares, Devin; Dulebohn, Daniel P; Svetlanov, Anton et al. (2013) Active and accurate trans-translation requires distinct determinants in the C-terminal tail of SmpB protein and the mRNA-like domain of transfer messenger RNA (tmRNA). J Biol Chem 288:30527-42
Guja, Kip E; Venkataraman, Krithika; Yakubovskaya, Elena et al. (2013) Structural basis for S-adenosylmethionine binding and methyltransferase activity by mitochondrial transcription factor B1. Nucleic Acids Res 41:7947-59
Svetlanov, Anton; Puri, Neha; Mena, Patricio et al. (2012) Francisella tularensis tmRNA system mutants are vulnerable to stress, avirulent in mice, and provide effective immune protection. Mol Microbiol 85:122-41
Mehta, Preeti; Woo, Perry; Venkataraman, Krithika et al. (2012) Ribosome purification approaches for studying interactions of regulatory proteins and RNAs with the ribosome. Methods Mol Biol 905:273-89
Srinivasan, Madhusudhan; Mehta, Preeti; Yu, Yao et al. (2011) The highly conserved KEOPS/EKC complex is essential for a universal tRNA modification, t6A. EMBO J 30:873-81
Ge, Zhiyun; Mehta, Preeti; Richards, Jamie et al. (2010) Non-stop mRNA decay initiates at the ribosome. Mol Microbiol 78:1159-70
Okan, Nihal A; Mena, Patricio; Benach, Jorge L et al. (2010) The smpB-ssrA mutant of Yersinia pestis functions as a live attenuated vaccine to protect mice against pulmonary plague infection. Infect Immun 78:1284-93

Showing the most recent 10 out of 14 publications