Programmed ribosomal frameshifting in decoding the SARS-CoV genome.
Programmed ribosomal frameshifting is an essential mechanism used for the expression of orf1b in coronaviruses. Comparative analysis of the frameshift region reveals a universal shift site U_UUA_AAC, followed by a predicted downstream RNA structure in the form of either a pseudoknot or kissing stem loops. Frameshifting in SARS-CoV has been characterized in cultured mammalian cells using a dual luciferase reporter system and mass spectrometry. Mutagenic analysis of the SARS-CoV shift site and mass spectrometry of an affinity tagged frameshift product confirmed tandem tRNA slippage on the sequence U_UUA_AAC. Analysis of the downstream pseudoknot stimulator of frameshifting in SARS-CoV shows that a proposed RNA secondary structure in loop II and two unpaired nucleotides at the stem I-stem II junction in SARS-CoV are important for frameshift stimulation. These results demonstrate key sequences required for efficient frameshifting, and the utility of mass spectrometry to study ribosomal frameshifting.
Baranov
Pavel V
PV
Department of Human Genetics, University of Utah, 15 N 2030 E, Room 7410, Salt Lake City, UT 84112-5330, USA.
Henderson
Clark M
CM
Anderson
Christine B
CB
Gesteland
Raymond F
RF
Atkins
John F
JF
Howard
Michael T
MT
D001483:Base Sequence; D002460:Cell Line; D017934:Coronavirus / Q000235:genetics*; D018965:Frameshifting, Ribosomal / Q000235:genetics*; D017930:Genes, Reporter; D016679:Genome, Viral; D006801:Humans; D008156:Luciferases / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012367:RNA, Viral / Q000737:chemistry / Q000235:genetics; D045473:Severe acute respiratory syndrome-related coronavirus / Q000235:genetics*; D016415:Sequence Alignment; D021241:Spectrometry, Mass, Electrospray Ionization
D012367:RNA, Viral; D008156:Luciferases