Abstract

Ribosomes translate the genetic information contained in mRNAs into protein by linking together amino acids with the help of aminoacyl-tRNAs. In bacteria, protein synthesis stalls when the ribosome reaches the 3'-end of truncated mRNA transcripts lacking a stop codon. Trans-translation is a conserved bacterial quality control process that rescues stalled ribosomes. Transfer-messenger RNA (tmRNA) and its protein partner SmpB mimic a tRNA by entering the A site of the ribosome and accepting the growing peptide chain. The ribosome releases the truncated mRNA and resumes translation on the tmRNA template. The open reading frame found on tmRNA encodes a peptide tag that marks the defective nascent peptide for proteolysis. A stop codon at the end of the open reading frame allows the ribosome to be recycled and engage in future rounds of translation.The entry of tmRNA into stalled ribosomes presents a challenge to our understanding of ribosome function because during the canonical decoding process, the ribosome specifically recognizes the codon-anticodon duplex formed between tRNA and mRNA in the A site. Recognition of proper base-pairing leads to conformational changes that accelerate GTP hydrolysis by EF-Tu and rapid accommodation of the tRNA into the ribosome for peptidyl transfer. The puzzle is that tmRNA enters stalled ribosomes and reacts with the nascent peptide in the absence of a codon-anticodon interaction. Instead, SmpB binding in the decoding center begins the rescue process, but it has been unclear how SmpB licenses tmRNA entry into stalled ribosomes. We analyzed a series of SmpB and ribosomal RNA mutants using pre-steady-state kinetic assays for EF-Tu activation and peptidyl transfer. Although the conserved 16S nucleotides A1492 and A1493 play an essential role in canonical decoding, they play little or no role in EF-Tu activation or peptidyl transfer to tmRNA. In contrast, a third nucleotide, G530, stacks with the side chain of SmpB residue His136, inducing conformational changes that lead to GTP hydrolysis by EF-Tu. A portion of the C-terminal tail forms a helix within the mRNA channel, monitoring the length of mRNA bound in the ribosome to avoid aborting productive protein synthesis. Helix formation in the mRNA channel is essential for accommodation and peptidyl transfer, but not for GTP hydrolysis. We show that conserved residues in the tail are essential for EF-Tu activation, accommodation, or translocation to the P site. Our findings lead to a clearer model of how the tmRNA-SmpB complex enters stalled ribosomes.

Degree

PhD

College and Department

Physical and Mathematical Sciences; Chemistry and Biochemistry

Rights

http://lib.byu.edu/about/copyright/

Date Submitted

2013-07-03

Document Type

Dissertation

Handle

http://hdl.lib.byu.edu/1877/etd6394

Keywords

tmRNA, SmpB, decoding, EF-Tu, ribosome

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