title: Identification of an essential pseudoknot in the putative downstream internal ribosome entry site in giardiavirus transcript.
abstract: Enhanced translation of giardiavirus-luciferase chimeric mRNA in Giardia lamblia requires the initial 264-nt viral capsid coding region as a putative internal ribosomal entry site (IRES). Essential structural elements in this site include (1) a downstream box (DB) complementary to the anti-DB at the 3' end of 16S-like rRNA, (2) stem-loops I, II, III, and IVA, and (3) a pentanucleotide 5'-UCUCC-3' immediately downstream from stem loop IVA. A search for the structural role of the pentanucleotide suggested that it may form a pseudoknot with another pentanucleotide 5'-GGAGA-3' in loop II. Alteration of the two pentanucleotides by site-directed mutagenesis resulted in a drastic reduction in translation of the transcript. But the loss was recovered by compensatory changes in the two sequences, suggesting Watson-Crick base pairings between them. Results from in vitro enzymatic and chemical structural probing supported the presence of such a pseudoknot 143 nt downstream from the initiation codon. Minor repositioning of this codon led invariably to a complete loss of translation, suggesting that the initiation site is confined within a rigid position defined by all the structural elements in the IRES including the pseudoknot. This is the first pseudoknot of its kind shown to play an important role in a downstream IRES of a viral transcript. The finding is particularly interesting because it could reflect a unique feature of translation initiation in Giardia, which is known to have exceedingly short (1-6 nt) 5' untranslated regions in its mRNAs.
authors.affiliation: Department of Pharmaceutical Chemistry, University of California, San Francisco 94143-0446, USA.
authors.forename: Srinivas
authors.initials: S
authors.lastname: Garlapati
authors.forename: Ching C
authors.initials: CC
authors.lastname: Wang
mesh_terms: D000818:Animals; D001483:Base Sequence; D018387:Codon, Initiator / Q000235:genetics; D016829:Giardia lamblia / Q000821:virology; D019198:Giardiavirus / Q000235:genetics*; D008156:Luciferases / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D012333:RNA, Messenger / Q000737:chemistry* / Q000235:genetics*; D012367:RNA, Viral / Q000737:chemistry* / Q000235:genetics*; D012260:Ribonucleases; D012270:Ribosomes / Q000235:genetics; D014158:Transcription, Genetic; D014162:Transfection
chemical_list: D018387:Codon, Initiator; D012333:RNA, Messenger; D012367:RNA, Viral; D008156:Luciferases; D012260:Ribonucleases