RRM
2006
Rustbelt RNA Meeting
RRM
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Poster abstracts
Abstract:
Formation of translation initiation complexes on leaderless mRNA occurs in the absence of signals upstream to the 5-terminal start codon. Primer extension inhibition assays (toeprints) have shown that 70S ribosomes from E. coli bind leaderless mRNA more strongly than 30S subunits in vitro, suggesting a novel mechanism for the formation of translation initiation complexes on leaderless mRNAs. In the current study, leaderless mRNA containing the nucleotide derivative 4-thiouridine (4S-U) is used in site-specific crosslinking assays to identify leaderless mRNA/ribosome interactions that occur during translation initiation. Photoactivation with ultraviolet light stimulates 4S-U to form covalent crosslinks to nearby nucleotides and amino acids. Gel shift assays have shown that an oligonucleotide containing the first 20 nucleotides of the bacteriophage lambda leaderless cI coding sequence, with 4S-U at the +2 position (i.e., the U of the AUG start codon), binds 70S ribosomes and 30S subunits from E. coli in a tRNA-dependent manner. Photoactivation of 4S-U in complexes containing 70S ribosomes or 30S subunits produced crosslinks to rRNA and r-proteins exclusively within the small subunit. Crosslinks of the leaderless mRNAs +2 position to 16S rRNA was localized to a single site at the 3 end close to position 1530. Crosslinked r-proteins were affinity selected and identified by MALDI-TOF mass spectrometry. Of the r-proteins identified, S1, S3, S7 and S10/S18 displayed specificity for a 5-AUG initiation codon. In accordance with the published crystal structure of the E. coli ribosome, it is likely that these r-proteins make contact with the 5-AUG before the formation of the codon/anti-codon interaction between the 5-AUG and initiator tRNA. This study will play a key role in understanding the mechanism by which these unique mRNAs are bound by ribosomes and translated into functional proteins.
Keywords: Translation Initiation, Ribosome, Leaderless mRNA
