Structure elucidation of the hepatitis B virus encapsidation signal by NMR on selectively labeled RNAs.
Hepatitis B virus (HBV) HBV is DNA virus with a unique replication strategy, which involves reverse transcription of its pregenomic RNA. Essential for this reverse transcription are the 5'- and 3'-ends of its pregenomic RNA (5'-RT-RNA and 3'-RT-RNA, respectively) which form conserved bulged stem-loop structures. The 5'-RT-RNA consists of a 67 nucleotide bulged stem-loop structure, epsilon, which constitutes the signal for encapsidation of the pregenomic RNA and subsequent reverse transcription. The reverse transcriptase (RT) initially binds to the completely conserved apical loop of epsilon and a 4-nucleotide primer is synthesized from the adjacent 6-nucleotide bulge. Structural studies of epsilon can provide important parameters required for the design of RNA targeted anti- viral drugs directed against Hepatitis B virus. NMR studies of large RNA systems (> ca. 50 nucleotides) require novel approaches, e.g., different labeling schemes and reduction of the system into separate structural building blocks. Recently, a new method of synthesizing (13)C/(15)N/(2)H labeled nucleotides has been developed based on converting specifically labeled glucose and bases into nucleotides by using enzymes from the pentose phosphate pathway and nucleotide and salvage pathways. These NTPs give a large freedom in designing different labeling patterns in in vitro synthesized RNAs under study for NMR. This opens up the way for NMR studies of RNAs that are considerably above the present size limit (up to 150 nucleotides). Here this new technique is applied for structural studies on 27, 36 and 61 nucleotides long RNA fragments, mimicking different regions of epsilon.
Flodell
Sara
S
Department of Medical Biosciences, Medical Biophysics, Umeå University, S-901 87 Umeå, Sweden.
Cromsigt
Jenny
J
Schleucher
Jürgen
J
Kidd-Ljunggren
Karin
K
Wijmenga
Sybren
S
D001483:Base Sequence; D002213:Capsid / Q000737:chemistry*; D004261:DNA Replication; D006515:Hepatitis B virus / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012367:RNA, Viral / Q000737:chemistry* / Q000235:genetics; D012194:RNA-Directed DNA Polymerase / Q000737:chemistry / Q000378:metabolism*
D012367:RNA, Viral; D012194:RNA-Directed DNA Polymerase