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

• Published in: RNA (Cambridge) Vol. 18; no. 3; pp. 508 - 518
• Main Authors: Giliberti, Jacqueline, O'Donnell, Sean, Etten, William J Van, Janssen, Gary R
• Format: Journal Article
• Language: English
• Published: United States Cold Spring Harbor Laboratory Press 01.03.2012
• Subjects: 5' Untranslated Regions; Dinucleoside Phosphates - metabolism; Escherichia coli; Escherichia coli - genetics; Escherichia coli - metabolism; Gene Fusion - genetics; Hydroxides - metabolism; Protein Biosynthesis; Ribosomes - metabolism; RNA, Bacterial - chemistry; RNA, Bacterial - metabolism; RNA, Catalytic - metabolism; RNA, Messenger - chemistry; RNA, Messenger - metabolism; Transcription, Genetic
• ISSN: 1355-8382; 1469-9001
• DOI: 10.1261/rna.027698.111

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.