RRM
2008 Rustbelt RNA Meeting
RRM
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Poster abstracts
Abstract:
Translation initiation in bacteria involves recruitment and binding of mRNA to ribosomes through a complementary Shine-Dalgarno (SD) – anti Shine-Dalgarno (ASD) interaction. We have added saturating amounts of a DNA oligonucleotide, complementary to the ribosome’s ASD sequence, to ribosomes, thereby eliminating its availability to the mRNA SD sequence. Despite having neutralized what is generally thought to be the primary mechanism for ribosome binding, the ribosomes still bound to the test mRNA. Data from primer extension inhibition (toeprint) assays suggests that blocking the SD-ASD interaction does not eliminate mRNA-ribosome binding and suggests that additional signals in the mRNA exist to promote ribosome binding. Additional evidence suggests that secondary signals in the mRNA not only promote ribosome binding but also allow translation. This was accomplished by mutating the SD sequence in the untranslated lac leader to its complementary nucleotide sequence, thereby eliminating its complementarity to the anti-SD found on the 30S ribosomal subunit. The lac leader region, both with and without SD mutations, was fused to a lacZ reporter gene and was assayed for the production of beta-galactosidase. Growth on plates containing X-gal indicated that presence of lacZ mRNA with the mutated SD still promoted translation initiation in E. coli. To further investigate the presence of additional signals, a series of deletions to the 5’UTR, both with and without the SD mutations, were fused to a lacZ reporter and transformed into E. coli. The constructs will be assayed for beta-galactosidase activity and for in vitro ribosome binding. The goal of this project is to identify the nucleotide sequences responsible for ribosome binding and expression in the absence of a SD-ASD interaction.
Keywords: ribosome binding, translation initiation, mRNA untranslated leader
