SUNY at Albany
June 19-23, 2001
The NMR Structure of the Reverse Transcriptase Binding Region of the Pregenomic RNA of the Hepatitis B Virus
Recently, a new methodology for the synthesis of 13C/15N/2H-labeled nucleotides (NTPs) has been developed based on the glucose pathway (1,2). Via this methodology NTPs can be produced with a variety of different labeling patterns. These NTPs give therefore a large freedom in designing different labeling schemes in enzymatically 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) (2,3). We are applying this new labeling methodology in the structure elucidation of the precore mRNA of the Hepatitis B virus (HBV) under study in our lab.
HBV is a virus with one of the smallest DNA-genomes (ca. 3.2kb). It has a unique replication strategy, which involves reverse transcription of its pregenomic RNA. The pregenomic RNA contains two similar conserved RNA structures located near its 5'-end (5'-RT-RNA) and 3'-end (3'-RT-RNA), respectively, that are essential for the reverse transcription. The 5'-RT-RNA contains a 60 nucleotide bulged stem-loop structure, called epsilon. The reverse transcriptase (RT) initially binds to the completely conserved apical stem-loop and a 4-nucleotide primer is synthesized from the adjacent 6-nucleotide bulge. The RT with its covalently bound primer subsequently transfers to the 3'-RT-RNA to start reverse transcription. The size of the two regions (150 to 200 nucleotides) is too large for present day NMR methods. Tackling such a system requires novel approaches such as the new labeling methodology described above and reduction of the system into separate structural building blocks. Here we focus on the important epsilon region.
Three RNA sequences covering different parts of epsilon were designed for NMR studies: 1) the apical stem-loop; 2) the bulged region; 3) the whole epsilon (61 nts). Each of these sequences fold into a stable structure as follows from (partial) assignment of the imino-spectra of unlabeled versions of these RNAs. Of the apical stem-loop, the sequential assignment of the non-exchangeable protons is essentially complete and various NOE connections have been derived. The loop that caps the stem does not form the predicted six-membered loop. Instead a smaller loop is formed. To improve on the low-resolution model (increase the number of assigned NOE contacts) labeling of this RNA according to the new labeling schemes is now in progress. This structure gives new clues regarding the conservation of the stem loop and might help in the understanding of the crucial priming step in reverse transcription.
References and Notes
Sara Flodell (1), Jenny Cromsigt (1), Hans Ippel (1), Jurgen Schleucher (1), Karin Kidd-Ljunggren (2) and Sybren Wijmenga (1)
(1)Dept. of Medical Biosciences, Medical Biophysics, Umea University, S-901 87 Umea, Sweden (2) Dept. of Infectious Diseases, University Hospital, S-221 85 Lund, Sweden Phone: +46-90-7866943, Fax: +46-90-136310, email: firstname.lastname@example.org