title: Intramolecular triplex potential sequence within a gene down regulates its expression in vivo.
abstract: Polypurine/polypyrimidine sequences have been shown to adopt intramolecular triple helix structures under torsional stress and/or at low pH. Such sequences have been observed within the the regulatory as well as the coding regions of several genes and the involvement of triple helical structure adopted by these sequences in transcriptional control has been speculated. Taking advantage of codon degeneracy we have engineered a 38 bp long intramolecular triple helix potential polypurine/polypyrimidine sequence motif between the 37th and 50th codons of beta-galactosidase gene in the plasmid pBluescriptIISK+ to investigate whether in vivo E.coli RNA polymerase would transcribe sequence motifs adopting triple helix structure, when present within the coding region of the gene. E.coli JM109 cells transformed with this construct pSBT1, exhibited 80% inhibition of beta-galactosidase expression compared to another construct pSBmT12 made using less preferred codons for identical amino acid sequence, but lacking the polypurine/polypyrimidine sequence motif. Truncated beta-galactosidase transcripts were observed for pSBT1 but not for pSBmT12. Here we report that a putative triple helix potential sequence within a gene can down regulate its expression by partially blocking the transcription elongation in vivo.
authors.affiliation: Molecular Biophysics Unit, Indian Institute of Science, Bangalore.
authors.forename: P S
authors.initials: PS
authors.lastname: Sarkar
authors.forename: S K
authors.initials: SK
authors.lastname: Brahmachari
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D015152:Blotting, Northern; D002738:Chloroquine; D015536:Down-Regulation; D015180:Electrophoresis, Gel, Two-Dimensional; D004926:Escherichia coli; D007700:Kinetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000235:genetics; D010957:Plasmids; D012045:Regulatory Sequences, Nucleic Acid; D014158:Transcription, Genetic; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
chemical_list: D009838:Oligodeoxyribonucleotides; D002738:Chloroquine; D001616:beta-Galactosidase