title: Translation initiation with GUC codon in the archaeon Halobacterium salinarum: implications for translation of leaderless mRNA and strict correlation between translation initiation and presence of mRNA.
abstract: We have investigated whether anticodon sequence mutant of an archaeal initiator tRNA can initiate protein synthesis using reporter genes carrying mutations in the initiation codon. Halobacterium salinarum was used as the model organism and the bacterio-opsin gene (bop), which encodes the precursor of the protein component of the purple membrane protein bacterio-opsin (Bop), was chosen as the reporter. We demonstrate that a CAU to GAC anticodon sequence mutant of Haloferax volcanii initiator tRNA can initiate Bop protein synthesis using GUC as the initiation codon in H. salinarum. We generated four mutant bop genes, each carrying the AUG to GUC initiation codon mutation, with or without a compensatory mutation to maintain a predicted stem-loop structure at the 5'-end of the bop mRNA, and with or without mutations to test translation initiation at a site corresponding to the amino terminus of mature bacterio-opsin. H. salinarum chromosomal recombinants containing these mutant genes were phenotypically Pum- (purple membrane negative). Upon transformation with a plasmid carrying the mutant initiator tRNA gene, only strains designed to maintain the bop mRNA stem-loop structure produced Bop and were phenotypically Pum+ as indicated by purple colony colour, and immunoblotting and spectral analysis of cell extracts. Thus GUC can serve as an initiation codon in archaea and the stem-loop structure in the bop mRNA is important for translation. Interestingly, for the same mutant mRNA, only transformants that produce Bop protein contain bop mRNA. These results suggest either a strong coupling between translation and mRNA stability or strong transcriptional polarity in H. salinarum.
authors.affiliation: Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
authors.forename: Gayathri
authors.initials: G
authors.lastname: Srinivasan
authors.forename: Mark P
authors.initials: MP
authors.lastname: Krebs
authors.forename: Uttam L
authors.initials: UL
authors.lastname: RajBhandary
mesh_terms: D020121:5' Untranslated Regions; D019843:Archaeal Proteins / Q000096:biosynthesis* / Q000235:genetics; D001436:Bacteriorhodopsins / Q000096:biosynthesis / Q000235:genetics; D001483:Base Sequence; D018387:Codon, Initiator / Q000235:genetics*; D019766:Genes, Archaeal; D017930:Genes, Reporter; D019617:Halobacterium salinarum / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D010442:Peptide Chain Initiation, Translational / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D019642:RNA, Archaeal / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism*; D012358:RNA, Transfer, Met / Q000235:genetics* / Q000378:metabolism
chemical_list: D020121:5' Untranslated Regions; D019843:Archaeal Proteins; D018387:Codon, Initiator; D019642:RNA, Archaeal; D012333:RNA, Messenger; D012358:RNA, Transfer, Met; D001436:Bacteriorhodopsins; C017827:bacterio-opsin