TY - JOUR
T1 - Trans insertion-splicing
T2 - Ribozyme-catalyzed insertion of targeted sequences into RNAs
AU - Johnson, Ashley K.
AU - Sinha, Joy
AU - Testa, Stephen M.
PY - 2005/8/9
Y1 - 2005/8/9
N2 - A group I intron-derived ribozyme from Pneumocystis carinii has been previously shown to bind an exogenous RNA substrate, splice out an internal segment, and then ligate the two ends back together (the trans excision-splicing reaction). We demonstrate that this same ribozyme can perform a trans insertion-splicing (TIS) reaction, where the ribozyme binds two exogenous RNA substrates and inserts one directly into the other. Reactions were optimized for both yield and rate, with optimum reactions carried out in 10 mM MgCl 2 for 2 h. Reaction products are stable, with no visible loss at extended times. The ribozyme recognizes the two substrates primarily through base pairing and requires an ωG on the ribozyme and an ωG on the sequence being inserted. We give evidence that the reaction mechanism is not the reverse of the trans excision-splicing reaction, but is composed of three steps, with intermediates attached to the ribozyme. Surprisingly, the internal guide sequence of the ribozyme is utilized to sequentially bind both substrates, forming independent P1 helices. This is an indication that ribozymes with essentially the native intron sequence can catalyze reactions significantly more dynamic and complex than self-splicing. The implications of group I intron-derived ribozymes being able to catalyze this unique reaction, and via this mechanism, are discussed.
AB - A group I intron-derived ribozyme from Pneumocystis carinii has been previously shown to bind an exogenous RNA substrate, splice out an internal segment, and then ligate the two ends back together (the trans excision-splicing reaction). We demonstrate that this same ribozyme can perform a trans insertion-splicing (TIS) reaction, where the ribozyme binds two exogenous RNA substrates and inserts one directly into the other. Reactions were optimized for both yield and rate, with optimum reactions carried out in 10 mM MgCl 2 for 2 h. Reaction products are stable, with no visible loss at extended times. The ribozyme recognizes the two substrates primarily through base pairing and requires an ωG on the ribozyme and an ωG on the sequence being inserted. We give evidence that the reaction mechanism is not the reverse of the trans excision-splicing reaction, but is composed of three steps, with intermediates attached to the ribozyme. Surprisingly, the internal guide sequence of the ribozyme is utilized to sequentially bind both substrates, forming independent P1 helices. This is an indication that ribozymes with essentially the native intron sequence can catalyze reactions significantly more dynamic and complex than self-splicing. The implications of group I intron-derived ribozymes being able to catalyze this unique reaction, and via this mechanism, are discussed.
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U2 - 10.1021/bi0504815
DO - 10.1021/bi0504815
M3 - Article
C2 - 16060679
AN - SCOPUS:23244442274
SN - 0006-2960
VL - 44
SP - 10702
EP - 10710
JO - Biochemistry
JF - Biochemistry
IS - 31
ER -