scholarly journals Base pairing between the 3' exon and an internal guide sequence increases 3' splice site specificity in the Tetrahymena self-splicing rRNA intron.

1990 ◽  
Vol 10 (6) ◽  
pp. 2960-2965 ◽  
Author(s):  
E R Suh ◽  
R B Waring
Keyword(s):  
Splice Site ◽  
Site Specificity ◽  
Group I Introns ◽  
Base Pairing ◽  
Group I ◽  
Guide Sequence ◽  
Compensatory Mutations ◽  
Self Splicing ◽  
Splice Site Usage ◽  
Selection Of

It has been proposed that recognition of the 3' splice site in many group I introns involves base pairing between the start of the 3' exon and a region of the intron known as the internal guide sequence (R. W. Davies, R. B. Waring, J. Ray, T. A. Brown, and C. Scazzocchio, Nature [London] 300:719-724, 1982). We have examined this hypothesis, using the self-splicing rRNA intron from Tetrahymena thermophila. Mutations in the 3' exon that weaken this proposed pairing increased use of a downstream cryptic 3' splice site. Compensatory mutations in the guide sequence that restore this pairing resulted in even stronger selection of the normal 3' splice site. These changes in 3' splice site usage were more pronounced in the background of a mutation (414A) which resulted in an adenine instead of a guanine being the last base of the intron. These results show that the proposed pairing (P10) plays an important role in ensuring that cryptic 3' splice sites are selected against. Surprisingly, the 414A mutation alone did not result in activation of the cryptic 3' splice site.

Base pairing between the 3' exon and an internal guide sequence increases 3' splice site specificity in the Tetrahymena self-splicing rRNA intron

1990 ◽  
Vol 10 (6) ◽  
pp. 2960-2965
Author(s):  
E R Suh ◽  
R B Waring
Keyword(s):  
Splice Site ◽  
Site Specificity ◽  
Group I Introns ◽  
Base Pairing ◽  
Group I ◽  
Guide Sequence ◽  
Compensatory Mutations ◽  
Self Splicing ◽  
Splice Site Usage ◽  
Selection Of

It has been proposed that recognition of the 3' splice site in many group I introns involves base pairing between the start of the 3' exon and a region of the intron known as the internal guide sequence (R. W. Davies, R. B. Waring, J. Ray, T. A. Brown, and C. Scazzocchio, Nature [London] 300:719-724, 1982). We have examined this hypothesis, using the self-splicing rRNA intron from Tetrahymena thermophila. Mutations in the 3' exon that weaken this proposed pairing increased use of a downstream cryptic 3' splice site. Compensatory mutations in the guide sequence that restore this pairing resulted in even stronger selection of the normal 3' splice site. These changes in 3' splice site usage were more pronounced in the background of a mutation (414A) which resulted in an adenine instead of a guanine being the last base of the intron. These results show that the proposed pairing (P10) plays an important role in ensuring that cryptic 3' splice sites are selected against. Surprisingly, the 414A mutation alone did not result in activation of the cryptic 3' splice site.

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.

Mechanism of recognition of the 5′ splice site in self-splicing group I introns

Nature ◽  
1986 ◽  
Vol 322 (6074) ◽  
pp. 86-89 ◽  
Author(s):  
Gian Garriga ◽  
Alan M. Lambowitz ◽  
Tan Inoue ◽  
Thomas R. Cech
Keyword(s):  
Splice Site ◽  
Group I Introns ◽  
Group I ◽  
Mechanism Of Recognition ◽  
Self Splicing

scholarly journals CBP2 protein promotes in vitro excision of a yeast mitochondrial group I intron.

1989 ◽  
Vol 9 (12) ◽  
pp. 5424-5433 ◽  
Author(s):  
A Gampel ◽  
M Nishikimi ◽  
A Tzagoloff
Keyword(s):  
Splice Site ◽  
Group I Intron ◽  
Nuclear Gene ◽  
Group I Introns ◽  
Wild Type ◽  
Group I ◽  
Mitochondrial Cytochrome B ◽  
High Concentrations ◽  
Self Splicing

The terminal intron (bI2) of the yeast mitochondrial cytochrome b gene is a group I intron capable of self-splicing in vitro at high concentrations of Mg2+. Excision of bI2 in vivo, however, requires a protein encoded by the nuclear gene CBP2. The CBP2 protein has been partially purified from wild-type yeast mitochondria and shown to promote splicing at physiological concentrations of Mg2+. The self-splicing and protein-dependent splicing reactions utilized a guanosine nucleoside cofactor, the hallmark of group I intron self-splicing reactions. Furthermore, mutations that abolished the autocatalytic activity of bI2 also blocked protein-dependent splicing. These results indicated that protein-dependent excision of bI2 is an RNA-catalyzed process involving the same two-step transesterification mechanism responsible for self-splicing of group I introns. We propose that the CBP2 protein binds to the bI2 precursor, thereby stabilizing the catalytically active structure of the RNA. The same or a similar RNA structure is probably induced by high concentrations of Mg2+ in the absence of protein. Binding of the CBP2 protein to the unspliced precursor was supported by the observation that the protein-dependent reaction was saturable by the wild-type precursor. Protein-dependent splicing was competitively inhibited by excised bI2 and by a splicing-defective precursor with a mutation in the 5' exon near the splice site but not by a splicing-defective precursor with a mutation in the core structure. Binding of the CBP2 protein to the precursor RNA had an effect on the 5' splice site helix, as evidenced by suppression of the interaction of an exogenous dinucleotide with the internal guide sequence.

Selection of the 3′-splice site in group I introns

FEBS Letters ◽  
1989 ◽  
Vol 250 (2) ◽  
pp. 129-133 ◽  
Author(s):  
John M. Burke
Keyword(s):  
Splice Site ◽  
Group I Introns ◽  
Group I ◽  
Selection Of

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.

CBP2 protein promotes in vitro excision of a yeast mitochondrial group I intron

1989 ◽  
Vol 9 (12) ◽  
pp. 5424-5433
Author(s):  
A Gampel ◽  
M Nishikimi ◽  
A Tzagoloff
Keyword(s):  
Splice Site ◽  
Group I Intron ◽  
Nuclear Gene ◽  
Group I Introns ◽  
Wild Type ◽  
Group I ◽  
Mitochondrial Cytochrome B ◽  
High Concentrations ◽  
Self Splicing

The terminal intron (bI2) of the yeast mitochondrial cytochrome b gene is a group I intron capable of self-splicing in vitro at high concentrations of Mg2+. Excision of bI2 in vivo, however, requires a protein encoded by the nuclear gene CBP2. The CBP2 protein has been partially purified from wild-type yeast mitochondria and shown to promote splicing at physiological concentrations of Mg2+. The self-splicing and protein-dependent splicing reactions utilized a guanosine nucleoside cofactor, the hallmark of group I intron self-splicing reactions. Furthermore, mutations that abolished the autocatalytic activity of bI2 also blocked protein-dependent splicing. These results indicated that protein-dependent excision of bI2 is an RNA-catalyzed process involving the same two-step transesterification mechanism responsible for self-splicing of group I introns. We propose that the CBP2 protein binds to the bI2 precursor, thereby stabilizing the catalytically active structure of the RNA. The same or a similar RNA structure is probably induced by high concentrations of Mg2+ in the absence of protein. Binding of the CBP2 protein to the unspliced precursor was supported by the observation that the protein-dependent reaction was saturable by the wild-type precursor. Protein-dependent splicing was competitively inhibited by excised bI2 and by a splicing-defective precursor with a mutation in the 5' exon near the splice site but not by a splicing-defective precursor with a mutation in the core structure. Binding of the CBP2 protein to the precursor RNA had an effect on the 5' splice site helix, as evidenced by suppression of the interaction of an exogenous dinucleotide with the internal guide sequence.

scholarly journals Mobile Self-Splicing Group I Introns from thepsbA Gene of Chlamydomonas reinhardtii: Highly Efficient Homing of an Exogenous Intron Containing Its Own Promoter

2001 ◽  
Vol 21 (10) ◽  
pp. 3472-3481 ◽  
Author(s):  
Obed W. Odom ◽  
Stephen P. Holloway ◽  
Nita N. Deshpande ◽  
Jaesung Lee ◽  
David L. Herrin
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Open Reading Frames ◽  
Group I Introns ◽  
Resistance Marker ◽  
Free Standing ◽  
Spectinomycin Resistance ◽  
Highly Efficient ◽  
Group I ◽  
Short Exon ◽  
Self Splicing

ABSTRACT Introns 2 and 4 of the psbA gene of Chlamydomonas reinhardtii chloroplasts (Cr.psbA2 andCr.psbA4, respectively) contain large free-standing open reading frames (ORFs). We used transformation of an intronless-psbA strain (IL) to test whether these introns undergo homing. Each intron, plus short exon sequences, was cloned into a chloroplast expression vector in both orientations and then cotransformed into IL along with a spectinomycin resistance marker (16Srrn). For Cr.psbA2, the sense construct gave nearly 100% cointegration of the intron whereas the antisense construct gave 0%, consistent with homing. For Cr.psbA4, however, both orientations produced highly efficient cointegration of the intron. Efficient cointegration of Cr.psbA4 also occurred when the intron was introduced as a restriction fragment lacking any known promoter. Deletion of most of the ORF, however, abolished cointegration of the intron, consistent with homing. TheCr.psbA4 constructs also contained a 3-(3,4-dichlorophenyl)-1,1-dimethylurea resistance marker in exon 5, which was always present when the intron integrated, thus demonstrating exon coconversion. Remarkably, primary selection for this marker gave >100-fold more transformants (>10,000/μg of DNA) than did the spectinomycin resistance marker. A trans homing assay was developed for Cr.psbA4; the ORF-minus intron integrated when the ORF was cotransformed on a separate plasmid. This assay was used to identify an intronic region between bp −88 and −194 (relative to the ORF) that stimulated homing and contained a possible bacterial (−10, −35)-type promoter. Primer extension analysis detected a transcript that could originate from this promoter. Thus, this mobile, self-splicing intron also contains its own promoter for ORF expression. The implications of these results for horizontal intron transfer and organelle transformation are discussed.

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.

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