Poster abstracts

Poster number 38 submitted by Jacqueline Giliberti

Effect of Initiation Factors on Leaderless mRNA Binding to Escherichia coli Ribosomes

Jacqueline Giliberti (Deptartment of Microbiology, Miami University), Jay E. Brock (Deptartment of Microbiology, Miami University), Gary R. Janssen (Deptartment of Microbiology, Miami University)

Abstract:
Bacterial mRNAs typically contain a 5’ untranslated region (5’ UTR) that includes a translation initiation region (TIR) which contains signals, such as the Shine-Dalgarno sequence and the spacer, that are important for 30S ribosomal subunit binding. Initiation factors (IFs) are thought to play a role in adjusting the initiation codon into the ribosomal P-site. “Leaderless” messages, in contrast, lack a 5’UTR and the traditional TIR. Current evidence suggests that translation initiation of leaderless mRNAs may follow a novel pathway involving 70S ribosomes. We are interested in the interactions that occur between leaderless mRNAs and the 30S subunit or 70S ribosome during translation initiation.
This study focuses on the role of IFs in ribosome binding of cI leaderless mRNA to 30S subunits or 70S ribosomes from Escherichia coli. Primer extension inhibition (toeprint) assays using 30S subunits suggest that IF2 increases stable binding while IF3 inhibits binding to cI mRNA. Alternatively, 70S ribosome binding is not altered by the addition of IFs. Filter binding allows for the measurement of less stable complexes; preliminary data suggests that IFs do not affect 30S or 70S association with cI mRNA.
To study interactions that occur between ribosomes and cI mRNA, a 20-nucleotide RNA oligomer that corresponds to the first 20-nucleotides of cI with a 4-thiouridine (4SU) at the +2 position of the start codon (cI-4SU), was used in crosslinking assays. UV-photoactivation of 4SU stimulates crosslinking to nearby nucleotides and amino acids. Crosslinks to 16S rRNA have been identified and localized close to 1530. Crosslinked ribosomal proteins have been identified by mass spectrometry and include S1, S3, S7, and S10/S18. These crosslinks were unaffected by the presence of IFs and/or initiator tRNA. Similar results were observed with 70S ribosomes. These mRNA/ribosome/IF interactions are important for understanding translation initiation of leaderless messages.

Keywords: translation, initiation factors