Mechanistic Investigations of a Ribozyme Derived from the Tetrahymena Group I Intron:  Insights into Catalysis and the Second Step of Self-Splicing†

Biochemistry ◽  
1996 ◽  
Vol 35 (18) ◽  
pp. 5796-5809 ◽  
Author(s):  
Rui Mei ◽  
Daniel Herschlag
Keyword(s):  
Group I Intron ◽  
Second Step ◽  
Group I ◽  
Mechanistic Investigations ◽  
Self Splicing

scholarly journals The conserved U.G pair in the 5' splice site duplex of a group I intron is required in the first but not the second step of self-splicing.

1989 ◽  
Vol 9 (9) ◽  
pp. 3657-3666 ◽  
Author(s):  
E T Barfod ◽  
T R Cech
Keyword(s):  
Splice Site ◽  
Group I Intron ◽  
Splice Site Mutation ◽  
Second Step ◽  
Site Mutation ◽  
Catalytic Rate Constant ◽  
Group I ◽  
Normal Site ◽  
Guide Sequence ◽  
Self Splicing

Group I self-splicing introns have a 5' splice site duplex (P1) that contains a single conserved base pair (U.G). The U is the last nucleotide of the 5' exon, and the G is part of the internal guide sequence within the intron. Using site-specific mutagenesis and analysis of the rate and accuracy of splicing of the Tetrahymena thermophila group I intron, we found that both the U and the G of the U.G pair are important for the first step of self-splicing (attack of GTP at the 5' splice site). Mutation of the U to a purine activated cryptic 5' splice sites in which a U.G pair was restored; this result emphasizes the preference for a U.G at the splice site. Nevertheless, some splicing persisted at the normal site after introduction of a purine, suggesting that position within the P1 helix is another determinant of 5' splice site choice. When the U was changed to a C, the accuracy of splicing was not affected, but the Km for GTP was increased by a factor of 15 and the catalytic rate constant was decreased by a factor of 7. Substitution of U.A, U.U, G.G, or A.G for the conserved U.G decreased the rate of splicing by an even greater amount. In contrast, mutation of the conserved G enhanced the second step of splicing, as evidenced by a trans-splicing assay. Furthermore, a free 5' exon ending in A or C instead of the conserved U underwent efficient ligation. Thus, unlike the remainder of the P1 helix, which functions in both the first and second steps of self-splicing, the conserved U.G appears to be important only for the first step.

The conserved U.G pair in the 5' splice site duplex of a group I intron is required in the first but not the second step of self-splicing

1989 ◽  
Vol 9 (9) ◽  
pp. 3657-3666
Author(s):  
E T Barfod ◽  
T R Cech
Keyword(s):  
Splice Site ◽  
Group I Intron ◽  
Splice Site Mutation ◽  
Second Step ◽  
Site Mutation ◽  
Catalytic Rate Constant ◽  
Group I ◽  
Normal Site ◽  
Guide Sequence ◽  
Self Splicing

Group I self-splicing introns have a 5' splice site duplex (P1) that contains a single conserved base pair (U.G). The U is the last nucleotide of the 5' exon, and the G is part of the internal guide sequence within the intron. Using site-specific mutagenesis and analysis of the rate and accuracy of splicing of the Tetrahymena thermophila group I intron, we found that both the U and the G of the U.G pair are important for the first step of self-splicing (attack of GTP at the 5' splice site). Mutation of the U to a purine activated cryptic 5' splice sites in which a U.G pair was restored; this result emphasizes the preference for a U.G at the splice site. Nevertheless, some splicing persisted at the normal site after introduction of a purine, suggesting that position within the P1 helix is another determinant of 5' splice site choice. When the U was changed to a C, the accuracy of splicing was not affected, but the Km for GTP was increased by a factor of 15 and the catalytic rate constant was decreased by a factor of 7. Substitution of U.A, U.U, G.G, or A.G for the conserved U.G decreased the rate of splicing by an even greater amount. In contrast, mutation of the conserved G enhanced the second step of splicing, as evidenced by a trans-splicing assay. Furthermore, a free 5' exon ending in A or C instead of the conserved U underwent efficient ligation. Thus, unlike the remainder of the P1 helix, which functions in both the first and second steps of self-splicing, the conserved U.G appears to be important only for the first step.

scholarly journals A Unique Group I Intron in Coxiella burnetii Is a Natural Splice Mutant

2009 ◽  
Vol 191 (12) ◽  
pp. 4044-4046 ◽  
Author(s):  
Rahul Raghavan ◽  
Linda D. Hicks ◽  
Michael F. Minnick
Keyword(s):  
Coxiella Burnetii ◽  
Group I Intron ◽  
23S Rrna ◽  
Rrna Gene ◽  
Group I Introns ◽  
Group I ◽  
23S Rrna Gene ◽  
Self Splicing

ABSTRACT Cbu.L1917, a group I intron present in the 23S rRNA gene of Coxiella burnetii, possesses a unique 3′-terminal adenine in place of a conserved guanine. Here, we show that, unlike all other group I introns, Cbu.L1917 utilizes a different cofactor for each splicing step and has a decreased self-splicing rate in vitro.

Crystal structure of a self-splicing group I intron with both exons

Nature ◽  
2004 ◽  
Vol 430 (6995) ◽  
pp. 45-50 ◽  
Author(s):  
Peter L. Adams ◽  
Mary R. Stahley ◽  
Anne B. Kosek ◽  
Jimin Wang ◽  
Scott A. Strobel
Keyword(s):  
Crystal Structure ◽  
Group I Intron ◽  
Group I ◽  
Self Splicing

scholarly journals A Self-Splicing Group I Intron in DNA Polymerase Genes of T7-Like Bacteriophages

2004 ◽  
Vol 186 (23) ◽  
pp. 8153-8155 ◽  
Author(s):  
Richard P. Bonocora ◽  
David A. Shub
Keyword(s):  
Dna Polymerase ◽  
Group I Intron ◽  
Group I Introns ◽  
Homing Endonucleases ◽  
Structural Element ◽  
Group I ◽  
Close Relatives ◽  
Self Splicing

ABSTRACT Group I introns are inserted into genes of a wide variety of bacteriophages of gram-positive bacteria. However, among the phages of enteric and other gram-negative proteobacteria, introns have been encountered only in phage T4 and several of its close relatives. Here we report the insertion of a self-splicing group I intron in the coding sequence of the DNA polymerase genes of ΦI and W31, phages that are closely related to T7. The introns belong to subgroup IA2 and both contain an open reading frame, inserted into structural element P6a, encoding a protein belonging to the HNH family of homing endonucleases. The introns splice efficiently in vivo and self-splice in vitro under mild conditions of ionic strength and temperature. We conclude that there is no barrier for maintenance of group I introns in phages of proteobacteria.

Hoechst 33258 Selectively Inhibits Group I Intron Self-Splicing by Affecting RNA Folding

ChemBioChem ◽  
2004 ◽  
Vol 5 (12) ◽  
pp. 1647-1652 ◽  
Author(s):  
Matthew D. Disney ◽  
Jessica L. Childs ◽  
Douglas H. Turner
Keyword(s):  
Rna Folding ◽  
Group I Intron ◽  
Hoechst 33258 ◽  
Group I ◽  
Self Splicing
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