The size of hammerhead ribozymes is related to cleavage kinetics: the role of substrate length.
The structure and function of small complexes formed between trans-cleaving hammerhead ribozymes and their substrates are being intensely studied in vitro. Conversely, target strands for hammerhead ribozymes in living cells are usually much longer, and cleavage kinetics in vitro of long substrates are usually approximately 100-fold slower. However, on the mechanistic level, not much is known about the influence of substrate length on cleavage kinetics. Here, we describe the influence of the length of the substrate strand on cleavage kinetics in vitro of two trans-cleaving hammerhead ribozymes. Progressive extension of the 3' end of the substrate decreases cleavage kinetics in a length-dependent manner. A six-nucleotide protruding 3' end of helix I is related to a decrease of the cleavage rate by one order of magnitude. Extension of the 5' end of the substrate shows a more complex relationship of the length-related decrease of cleavage activity. Decreased cleavage activity can be compensated by increased magnesium concentrations. An explanation of this finding does not seem to include major influences of the extended substrate on the thermal stability or the global structural arrangement of the three double-strand helices of the hammerhead structure. We hypothesize that long-range influences between the termini of the substrate strand and the catalytic centre could be responsible for decreased cleavage rates.
Hormes
Robert
R
Forschungsschwerpunkt Angewandte Tumorvirologie, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 242, 69120 Heidelberg, Germany.
Sczakiel
Georg
G
D000255:Adenosine Triphosphate / Q000378:metabolism; D001345:Autoradiography; D001483:Base Sequence; D015497:HIV-1 / Q000235:genetics; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000096:biosynthesis / Q000737:chemistry; D010759:Phosphorus Isotopes; D011533:Proviruses / Q000235:genetics; D016337:RNA, Catalytic / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D009841:Oligonucleotides; D010759:Phosphorus Isotopes; D016337:RNA, Catalytic; C427204:hammerhead ribozyme; D000255:Adenosine Triphosphate; D008274:Magnesium