title: A 5'-terminal phosphate is required for stable ternary complex formation and translation of leaderless mRNA in Escherichia coli.
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.
authors.affiliation: Affymetrix, Cleveland, Ohio 44128, USA.
authors.forename: Jacqueline
authors.initials: J
authors.lastname: Giliberti
authors.forename: Sean
authors.initials: S
authors.lastname: O'Donnell
authors.forename: William J Van
authors.initials: WJ
authors.lastname: Etten
authors.forename: Gary R
authors.initials: GR
authors.lastname: Janssen
mesh_terms: D020121:5' Untranslated Regions; D015226:Dinucleoside Phosphates / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D050939:Gene Fusion / Q000235:genetics; D006878:Hydroxides / Q000378:metabolism; D014176:Protein Biosynthesis; D012329:RNA, Bacterial / Q000737:chemistry / Q000378:metabolism; D016337:RNA, Catalytic / Q000378:metabolism; D012333:RNA, Messenger / Q000737:chemistry* / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism; D014158:Transcription, Genetic
chemical_list: D020121:5' Untranslated Regions; D015226:Dinucleoside Phosphates; D006878:Hydroxides; D012329:RNA, Bacterial; D016337:RNA, Catalytic; D012333:RNA, Messenger; C427204:hammerhead ribozyme; C031356:hydroxide ion