RRM
2006
Rustbelt RNA Meeting
RRM
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Poster abstracts
Abstract:
The E. coli aroL gene, encoding shikimate kinase II, contains an untranslated leader of 124 nucleotides (nts) that includes a canonical Shine-Dalgarno (SD) sequence positioned appropriately upstream to the aroL start codon. Ribosome binding studies (i.e., toeprint assays) revealed 30S ribosomal subunits bind at the aroL start codon and to one of two, or both, AUG triplet(s) located within the untranslated leader, upstream to the aroL start codon. Neither upstream start codon contains a SD sequence. AUG (number 1) is at position –67 relative to the aroL start codon and contains an in-frame stop codon nine nts away. The open reading frame specified by AUG (number 2), at position –62, encodes a putative peptide of 21 amino acids. The stop codon (UGA) in frame with AUG (number 2) overlaps the aroL start codon, suggesting aroL could be translationally coupled to the upstream AUG (number 2) open reading frame. Translational fusion of the AUG (number 1) open reading frame to a lacZ reporter gene suggests that it has minimal activity for initiating translation. Fusion of the AUG (number 2) reading frame to lacZ reveals a significant amount of translational activity, producing approximately 30 percent of the LacZ activity measured when the aroL coding sequence is fused to lacZ. Site-directed mutation of the AUG (number 1) had negligible effect on translation from the downstream aroL start codon whereas mutation of AUG (number 2) reduced expression from aroL by approximately 30 percent. Identification of the signals used by ribosomes to bind AUG (number 2), in the absence of a SD sequence, might reveal novel signals used by ribosomes to find and bind translational start sites in other mRNAs.
Keywords: Translational Coupling, Ribosome Binding, untranslated leader
