Book of Abstracts: Albany 2003

category image Albany 2003
Conversation 13
Abstract Book
June 17-21 2003

Visualizing tmRNA Entry into the Stalled Ribosome

Bacterial ribosomes stalled on defective messenger RNAs (mRNAs) are rescued by tmRNA, a ~300 nucleotide long molecule that functions as both transfer RNA (tRNA) and mRNA. Translation then switches from the defective mRNA to a short open reading frame (ORF) on tmRNA that tags the defective nascent peptide chain for degradation. However, the mechanism by which tmRNA can enter and move through the ribosome is unknown. We present a cryo-electron microscopy study at ~13-15Å showing the entry of tmRNA from Thermus thermophilus in a complex with EF-Tu·GDP, stalled with the antibiotic kirromycin, into the ribosome. Apart from EF-Tu, the tmRNA interaction with the ribosome requires two other proteins; a small protein B (SmpB), which has been shown to be required for tmRNA-mediated peptide-tagging activity, and ribosomal protein S1, which is known to bind single-stranded RNA and has been proposed to be required for tmRNA binding to the ribosome. The cryo-EM structure gives clues on how tmRNA could move through the ribosome despite its complicated topology and addresses a number of interesting questions regarding the way tmRNA functions. It shows that EF-Tu brings aminoacylated tmRNA to the ribosome in a manner similar to canonical tRNAs. The protein SmpB bridges tmRNA and the 50S subunit. A mimicry of codon-anticodon base pairing between distal parts of tmRNA is shown to be unlikely; rather, an internal loop between helices 2a and 2b is situated close to the decoding site. Protein S1 is potentially involved in unwinding a portion of the ORF. The cryo-EM structure reveals the locations of the pseudoknots and sheds some light on their individual roles.

Sukhjit Kaur2
Mikel Valle1
Reynald Gillet3
Anke Henne4
V. Ramakrishnan3
Joachim Frank1, 2, 5

1Howard Hughes Medical Institute
2Health Research, Inc.
Wadsworth Center
Empire State Plaza
Albany, NY 12201-0509, USA
Phone: (518) 474 2253
Fax: (518) 486 2191
3MRC Laboratory of Molecular Biology
Hills Road
Cambridge CB2 2QH
United Kingdom
4Göttingen Genomics Laboratory
Institute of Microbiology and Genetics
Grisebachstr. 8
37077 Göttingen, Germany
5Department of Biomedical Sciences
State University of New York at Albany
Empire State Plaza
Albany, New York 12201-0509