Grant: $196,875 - National Institutes of Health - Jul. 30, 2009
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Award Description: The mechanism of tmRNA entry into stalled ribosomes tmRNA rescues ribosomes stalled in bacteria. Mimicking a transfer RNA, tmRNA enters stalled ribosomes and transfers Ala to the growing peptide chain. The ribosome then resumes translation with tmRNA as a template, adding a ten amino acid sequence that tags the nascent polypeptide for proteolysis. How are tmRNA and its protein partner SmpB accepted into stalled ribosomes? During normal translation, robust decoding mechanisms ensure that ribosomes select the proper, cognate tRNAs. 16S ribosomal RNA nucleotides G530, A1492, and A1493 monitor the codon-anticodon interaction. When they detect the proper pairing geometry, a conformational change occurs in the decoding center, the 30S subunit assumes a closed form, and EF-Tu hydrolyzes GTP. The aminoacylated 3’-end of the tRNA is then accommodated into the peptidyl-transferase center where the new peptide bond is rapidly formed. Since tmRNA is not able to participate in codon-anticodon pairing, how does it get past this decoding machinery? The SmpB protein binds to the decoding center; its C-terminal tail somehow activates EF-Tu and promotes tmRNA accommodation. The objective of this proposal is to determine the molecular mechanism by which this occurs. Pre-steady state kinetics experiments will be performed to measure the rates of EF-Tu activation and tmRNA accommodation for a series of SmpB, tmRNA, and ribosome mutants. These studies will identify the residues in each molecule that act to promote decoding at rapid rates. The specific aims of the project are: 1) Determine if the SmpB tail acts as a helix within the ribosome. 2) Determine the role of key conserved amino acids in the SmpB tail. 3) Analyze the role of 16S nucleotides G530, A1492, and A1493 in the SmpB-induced decoding process. 4) Determine if SmpB and tmRNA play an active role in transmitting the decoding signal.
Project Description: The project start date was August 3, 2009. These supplemental funds were used to support travel to Baltimore, MD, for a graduate research assistant. He spent one week in the lab of our collaborator, Rachel Green, at the Johns Hopkins School of Medicine. He performed kinetic analysis of the reaction of tmRNA and SmpB with stalled ribosomes. Peptide-bond formation rates were measured for a series of SmpB mutants and ribosome mutants. His data show that several ribosomal nucleotides required for normal decoding (with normal tRNAs) do not play a role in decoding with tmRNA and SmpB. He identified several SmpB mutants with lower rates, identifying key residues in SmpB essential for decoding. These mutations are found in the SmpB C-terminal tail. His work also shows that the C-terminal tail forms a helix within the ribosome for decoding to occur. A manuscript combining these findings with in vivo characterization of the system is currently in preparation. This constitutes progress towards the first three out of four specific aims for this grant.
Jobs Summary: One new Graduate Research Assistant position was created (Total jobs reported: 0)
Project Status: Less Than 50% Completed
This award's data was last updated on Jul. 30, 2009. Help expand these official descriptions using the wiki below.