title: Investigating the in vivo activity of the DeaD protein using protein-protein interactions and the translational activity of structured chloramphenicol acetyltransferase mRNAs.
abstract: Here, we report the use of an in vivo protein-protein interaction detection approach together with focused follow-up experiments to study the function of the DeaD protein in Escherichia coli. In this method, functions are assigned to proteins based on the interactions they make with others in the living cell. The assigned functions are further confirmed using follow-up experiments. The DeaD protein has been characterized in vitro as a putative prokaryotic factor required for the formation of translation initiation complexes on structured mRNAs. Although the RNA helicase activity of DeaD has been demonstrated in vitro, its in vivo activity remains controversial. Here, using a method called sequential peptide affinity (SPA) tagging, we show that DeaD interacts with certain ribosomal proteins as well as a series of other nucleic acid binding proteins. Focused follow-up experiments provide evidence for the mRNA helicase activity of the DeaD protein complex during translation initiation. DeaD overexpression compensates for the reduction of the translation activity caused by a structure placed at the initiation region of a chloramphenicol acetyltransferase gene (cat) used as a reporter. Deletion of the deaD gene, encoding DeaD, abolishes the translation activity of the mRNA with an inhibitory structure at its initiation region. Increasing the growth temperature disrupts RNA secondary structures and bypasses the DeaD requirement. These observations suggest that DeaD is involved in destabilizing mRNA structures during translation initiation. This study also provides further confirmation that large-scale protein-protein interaction data can be suitable to study protein functions in E. coli.
authors.affiliation: Department of Medical Genetics and Microbiology, Banting and Best Institute of Medical Research, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Gareth
authors.initials: G
authors.lastname: Butland
authors.forename: Nevan J
authors.initials: NJ
authors.lastname: Krogan
authors.forename: Jianhua
authors.initials: J
authors.lastname: Xu
authors.forename: Wen-Hong
authors.initials: WH
authors.lastname: Yang
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Aoki
authors.forename: Joyce S
authors.initials: JS
authors.lastname: Li
authors.forename: Naden
authors.initials: N
authors.lastname: Krogan
authors.forename: Javier
authors.initials: J
authors.lastname: Menendez
authors.forename: Gerard
authors.initials: G
authors.lastname: Cagney
authors.forename: Gholam C
authors.initials: GC
authors.lastname: Kiani
authors.forename: Mathew G
authors.initials: MG
authors.lastname: Jessulat
authors.forename: Nira
authors.initials: N
authors.lastname: Datta
authors.forename: Ivan
authors.initials: I
authors.lastname: Ivanov
authors.forename: Mounir G
authors.initials: MG
authors.lastname: Abouhaidar
authors.forename: Andrew
authors.initials: A
authors.lastname: Emili
authors.forename: Jack
authors.initials: J
authors.lastname: Greenblatt
authors.forename: M Clelia
authors.initials: MC
authors.lastname: Ganoza
authors.forename: Ashkan
authors.initials: A
authors.lastname: Golshani
mesh_terms: D001483:Base Sequence; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics*; D053487:DEAD-box RNA Helicases / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics*
chemical_list: D017931:DNA Primers; D029968:Escherichia coli Proteins; D012333:RNA, Messenger; D015500:Chloramphenicol O-Acetyltransferase; C088840:deaD protein, E coli; D053487:DEAD-box RNA Helicases