A 5′-terminal phosphate is required for stable ternary complex formation and translation of leaderless mRNA in Escherichia coli

Jacqueline Giliberti; Sean O'Donnell; William J. Van Etten; Gary R. Janssen

doi:10.1261/rna.027698.111

A 5′-terminal phosphate is required for stable ternary complex formation and translation of leaderless mRNA in Escherichia coli

¹Affymetrix, Cleveland, Ohio 44128, USA
²Grifols, Inc., Research Triangle Park, North Carolina 27709, USA
³BioTeam Inc., Middleton, Massachusetts 01949, USA
⁴Department of Microbiology, Miami University, Oxford, Ohio 45056, USA

Abstract

The bacteriophage λ's cI mRNA was utilized to examine the importance of the 5′-terminal phosphate on expression of leadered and leaderless mRNA in Escherichia coli. A hammerhead ribozyme was used to produce leadered and leaderless mRNAs, in vivo and in vitro, that contain a 5′-hydroxyl. Although these mRNAs may not occur naturally in the bacterial cell, they allow for the study of the importance of the 5′-phosphorylation state in ribosome binding and translation of leadered and leaderless mRNAs. Analyses with mRNAs containing either a 5′-phosphate or a 5′-hydroxyl indicate that leaderless cI mRNA requires a 5′-phosphate for stable ribosome binding in vitro as well as expression in vivo. Ribosome-binding assays show that 30S subunits and 70S ribosomes do not bind as strongly to 5′-hydroxyl as they do to 5′-phosphate containing leaderless mRNA and the tRNA-dependent ternary complex is less stable. Additionally, filter-binding assays revealed that the 70S ternary complex formed with a leaderless mRNA containing a 5′-hydroxyl has a dissociation rate (k_off) that is 4.5-fold higher compared with the complex formed with a 5′-phosphate leaderless mRNA. Fusion to a lacZ reporter gene revealed that leaderless cI mRNA expression with a 5′-hydroxyl was >100-fold lower than the equivalent mRNA with a 5′-phosphate. These data indicate that a 5′-phosphate is an important feature of leaderless mRNA for stable ribosome binding and expression.

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Footnotes

↵5 Corresponding author.

E-mail janssegr{at}muohio.edu.
Article published online ahead of print. Article and publication date are at http://www.rnajournal.org/cgi/doi/10.1261/rna.027698.111.