The poly(A) site sequence in HDV RNA alters both extent and rate of self-cleavage of the antigenomic ribozyme.
The ribozyme self-cleavage site in the antigenomic sequence of hepatitis delta virus (HDV) RNA is 33-nt downstream of the poly(A) site for the delta antigen mRNA. An HDV antigenomic ribozyme precursor RNA that included the upstream poly(A) processing site was used to test the hypothesis that nonribozyme sequence near the poly(A) site could affect ribozyme activity. Relative to ribozyme precursor without the extra upstream sequences, the kinetic profile for self-cleavage of the longer precursor was altered in two ways. First, only half of the precursor RNA self-cleaved. The cleaved fraction could be increased or decreased with mutations in the upstream sequence. These mutations, which were predicted to alter the relative stability of competing secondary structures within the precursor, changed the distribution of alternative RNA structures that are resolved in native-gel electrophoresis. Second, the active fraction cleaved with an observed rate constant that was higher than that of the ribozyme without the upstream sequences. Moreover, the higher rate constants occurred at lower, near-physiological, divalent metal ion concentrations (1-2 mM). Modulation of ribozyme activity, through competing alternative structures, could be part of a mechanism that allows a biologically significant choice between maturation of the mRNA and processing of replication intermediates.
Brown
Abigail L
AL
Department of Biochemistry, Duke University Medical Center, Durham NC 27710, USA.
Perrotta
Anne T
AT
Wadkins
Timothy S
TS
Been
Michael D
MD
D020121:5' Untranslated Regions / Q000737:chemistry; D020029:Base Pairing; D001483:Base Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D003698:Hepatitis Delta Virus / Q000201:enzymology / Q000235:genetics*; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011061:Poly A / Q000737:chemistry*; D012322:RNA Precursors / Q000737:chemistry; D016337:RNA, Catalytic / Q000737:chemistry* / Q000378:metabolism; D012367:RNA, Viral / Q000737:chemistry* / Q000378:metabolism
D020121:5' Untranslated Regions; D012322:RNA Precursors; D016337:RNA, Catalytic; D012367:RNA, Viral; D011061:Poly A; D008274:Magnesium