title: In vivo analysis of translation initiation sites in Plasmodium falciparum.
abstract: Regulation of gene expression in the malaria parasite Plasmodium falciparum is tightly controlled and little is known about the many steps involved. One step i.e. translation initiation is also poorly understood and in P. falciparum, choice of the translation initiation site (TIS) is a critical decision largely due to the high frequency of AUGs in the relatively long 5' untranslated regions of parasite mRNAs. The sequences surrounding the TIS have a major role to play in translation initiation and this report evaluates these sequences by mutational analysis of the heat shock protein 86 gene, transient transfection and reporter assays in the parasite. We find that purines at the -3 and +4 positions are essential for efficient translation in P. falciparum, similar to other eukaryotes. Interestingly, a U at the -1 position results in 2.5-fold higher reporter activity compared to wild type. Certain classes of protein biosynthetic genes show higher frequencies of U at the -1 position, suggesting that these genes may exhibit higher levels of translation. This work defines the optimal sequences for TIS choice and has implications for the design of efficient expression vectors in an important human pathogen.
authors.affiliation: Department of Biosciences & Bioengineering, Indian Institute of Technology Bombay, Powai, Mumbai 400076, India.
authors.forename: Balakota Reddy
authors.initials: BR
authors.lastname: Patakottu
authors.forename: Prashant Kumar
authors.initials: PK
authors.lastname: Singh
authors.forename: Pawan
authors.initials: P
authors.lastname: Malhotra
authors.forename: V S
authors.initials: VS
authors.lastname: Chauhan
authors.forename: Swati
authors.initials: S
authors.lastname: Patankar
mesh_terms: D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D005786:Gene Expression Regulation / Q000235:genetics / Q000502:physiology*; D005822:Genetic Vectors / Q000235:genetics; D018841:HSP90 Heat-Shock Proteins / Q000235:genetics*; D008156:Luciferases; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010442:Peptide Chain Initiation, Translational / Q000235:genetics*; D010963:Plasmodium falciparum / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics*; D017422:Sequence Analysis, DNA
chemical_list: D017931:DNA Primers; D018841:HSP90 Heat-Shock Proteins; D012333:RNA, Messenger; D008156:Luciferases