title: Distinct tmRNA sequence elements facilitate RNase R engagement on rescued ribosomes for selective nonstop mRNA decay.
abstract: trans-Translation, orchestrated by SmpB and tmRNA, is the principal eubacterial pathway for resolving stalled translation complexes. RNase R, the leading nonstop mRNA surveillance factor, is recruited to stalled ribosomes in a trans-translation dependent process. To elucidate the contributions of SmpB and tmRNA to RNase R recruitment, we evaluated Escherichia coli-Francisella tularensis chimeric variants of tmRNA and SmpB. This evaluation showed that while the hybrid tmRNA supported nascent polypeptide tagging and ribosome rescue, it suffered defects in facilitating RNase R recruitment to stalled ribosomes. To gain further insights, we used established tmRNA and SmpB variants that impact distinct stages of the trans-translation process. Analysis of select tmRNA variants revealed that the sequence composition and positioning of the ultimate and penultimate codons of the tmRNA ORF play a crucial role in recruiting RNase R to rescued ribosomes. Evaluation of defined SmpB C-terminal tail variants highlighted the importance of establishing the tmRNA reading frame, and provided valuable clues into the timing of RNase R recruitment to rescued ribosomes. Taken together, these studies demonstrate that productive RNase R-ribosomes engagement requires active trans-translation, and suggest that RNase R captures the emerging nonstop mRNA at an early stage after establishment of the tmRNA ORF as the surrogate mRNA template.
authors.affiliation: Department of Biochemistry and Cell Biology, Center for Infectious Diseases, Stony Brook University, Stony Brook, New York, NY 11794, USA.
authors.forename: Krithika
authors.initials: K
authors.lastname: Venkataraman
authors.affiliation: Department of Biochemistry and Cell Biology, Center for Infectious Diseases, Stony Brook University, Stony Brook, New York, NY 11794, USA.
authors.forename: Hina
authors.initials: H
authors.lastname: Zafar
authors.affiliation: Department of Biochemistry and Cell Biology, Center for Infectious Diseases, Stony Brook University, Stony Brook, New York, NY 11794, USA wali.karzai@stonybrook.edu.
authors.forename: A Wali
authors.initials: AW
authors.lastname: Karzai
mesh_terms: D004926:Escherichia coli / Q000235:genetics; D005095:Exoribonucleases / Q000378:metabolism*; D005604:Francisella tularensis / Q000235:genetics; D016366:Open Reading Frames; D014176:Protein Biosynthesis; D020871:RNA Stability; D012329:RNA, Bacterial / Q000737:chemistry* / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D038621:Regulatory Sequences, Ribonucleic Acid; D012270:Ribosomes / Q000201:enzymology / Q000378:metabolism*
chemical_list: D012329:RNA, Bacterial; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D038621:Regulatory Sequences, Ribonucleic Acid; C430328:small protein B; C096222:tmRNA; D005095:Exoribonucleases; C080868:ribonuclease R