scholarly journals Miniribozymes, small derivatives of the sunY intron, are catalytically active.

1989 ◽  
Vol 9 (12) ◽  
pp. 5480-5483 ◽  
Author(s):  
J A Doudna ◽  
J W Szostak
Keyword(s):  
Bacteriophage T4 ◽  
Complete Loss ◽  
The Self ◽  
Group I Introns ◽  
Structural Requirements ◽  
Group I ◽  
Catalytically Active ◽  
Conserved Core ◽  
Self Splicing ◽  
Derivatives Of

The self-splicing sunY intron from bacteriophage T4 has the smallest conserved core secondary structure of any of the active group I introns. Here we show that several nonconserved regions can be deleted from this intron without complete loss of catalytic activity. The 3' stems P9, P9.1, and P9.2 can be deleted while retaining 5' cleaving activity. Two base-paired stems (P7.1 and P7.2) that are peculiar to the group IA introns can also be deleted; however, the activities of the resulting derivatives depend greatly on the choice of replacement sequences and their lengths. The smallest active derivative is less than 180 nucleotides long. These experiments help to define the minimum structural requirements for catalysis.

Miniribozymes, small derivatives of the sunY intron, are catalytically active

1989 ◽  
Vol 9 (12) ◽  
pp. 5480-5483
Author(s):  
J A Doudna ◽  
J W Szostak
Keyword(s):  
Bacteriophage T4 ◽  
Complete Loss ◽  
The Self ◽  
Group I Introns ◽  
Structural Requirements ◽  
Group I ◽  
Catalytically Active ◽  
Conserved Core ◽  
Self Splicing ◽  
Derivatives Of

The self-splicing sunY intron from bacteriophage T4 has the smallest conserved core secondary structure of any of the active group I introns. Here we show that several nonconserved regions can be deleted from this intron without complete loss of catalytic activity. The 3' stems P9, P9.1, and P9.2 can be deleted while retaining 5' cleaving activity. Two base-paired stems (P7.1 and P7.2) that are peculiar to the group IA introns can also be deleted; however, the activities of the resulting derivatives depend greatly on the choice of replacement sequences and their lengths. The smallest active derivative is less than 180 nucleotides long. These experiments help to define the minimum structural requirements for catalysis.

scholarly journals Evaluating Group I Intron Catalytic Efficiency in Mammalian Cells

1999 ◽  
Vol 19 (10) ◽  
pp. 6479-6487 ◽  
Author(s):  
Meredith B. Long ◽  
Bruce A. Sullenger
Keyword(s):  
Rna Polymerase Ii ◽  
Mammalian Cells ◽  
Catalytic Efficiency ◽  
Human Cells ◽  
The Self ◽  
Group I Introns ◽  
Cellular Environment ◽  
Group I ◽  
Self Splicing ◽  
The Impact

ABSTRACT Recent reports have demonstrated that the group I ribozyme fromTetrahymena thermophila can performtrans-splicing reactions to repair mutant RNAs. For therapeutic use, such ribozymes must function efficiently when transcribed from genes delivered to human cells, yet it is unclear how group I splicing reactions are influenced by intracellular expression of the ribozyme. Here we evaluate the self-splicing efficiency of group I introns from transcripts expressed by RNA polymerase II in human cells to directly measure ribozyme catalysis in a therapeutically relevant setting. Intron-containing expression cassettes were transfected into a human cell line, and RNA transcripts were analyzed for intron removal. The percentage of transcripts that underwent self-splicing ranged from 0 to 50%, depending on the construct being tested. Thus, self-splicing activity is supported in the mammalian cellular environment. However, we find that the extent of self-splicing is greatly influenced by sequences flanking the intron and presumably reflects differences in the intron’s ability to fold into an active conformation inside the cell. In support of this hypothesis, we show that the ability of the intron to fold and self-splice from cellular transcripts in vitro correlates well with the catalytic efficiency observed from the same transcripts expressed inside cells. These results underscore the importance of evaluating the impact of sequence context on the activity of therapeutic group I ribozymes. The self-splicing system that we describe should facilitate these efforts as well as aid in efforts at enhancing in vivo ribozyme activity for various applications of RNA repair.

scholarly journals Self-Splicing of the Bacteriophage T4 Group I Introns Requires Efficient Translation of the Pre-mRNA In Vivo and Correlates with the Growth State of the Infected Bacterium

2006 ◽  
Vol 189 (3) ◽  
pp. 980-990 ◽  
Author(s):  
Linus Sandegren ◽  
Britt-Marie Sjöberg
Keyword(s):  
Stationary Phase ◽  
De Novo ◽  
Bacteriophage T4 ◽  
Group I Introns ◽  
Growth State ◽  
Group I ◽  
Splicing Efficiency ◽  
Self Splicing ◽  
Upstream Exon

ABSTRACT Bacteriophage T4 contains three self-splicing group I introns in genes in de novo deoxyribonucleotide biosynthesis (in td, coding for thymidylate synthase and in nrdB and nrdD, coding for ribonucleotide reductase). Their presence in these genes has fueled speculations that the introns are retained within the phage genome due to a possible regulatory role in the control of de novo deoxyribonucleotide synthesis. To study whether sequences in the upstream exon interfere with proper intron folding and splicing, we inhibited translation in T4-infected bacteria as well as in bacteria containing recombinant plasmids carrying the nrdB intron. Splicing was strongly reduced for all three T4 introns after the addition of chloramphenicol during phage infection, suggesting that the need for translating ribosomes is a general trait for unperturbed splicing. The splicing of the cloned nrdB intron was markedly reduced in the presence of chloramphenicol or when translation was hindered by stop codons inserted in the upstream exon. Several exon regions capable of forming putative interactions with nrdB intron sequences were identified, and the removal or mutation of these exon regions restored splicing efficiency in the absence of translation. Interestingly, splicing of the cloned nrdB intron was also reduced as cells entered stationary phase and splicing of all three introns was reduced upon the T4 infection of stationary-phase bacteria. Our results imply that conditions likely to be frequently encountered by natural phage populations may limit the self-splicing efficiency of group I introns. This is the first time that environmental effects on bacterial growth have been linked to the regulation of splicing of phage introns.

Modeling Study on the Cleavage Step of the Self-Splicing Reaction in Group I Introns

1993 ◽  
Vol 10 (6) ◽  
pp. 945-972 ◽  
Author(s):  
Robert F. Setlik ◽  
Ramon Garduno-Juarez ◽  
John I. Manchester ◽  
Masayuki Shibata ◽  
Rick L. Ornstein ◽  
...  
Keyword(s):  
The Self ◽  
Group I Introns ◽  
Group I ◽  
Cleavage Step ◽  
Self Splicing ◽  
Modeling Study

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.

Three-dimensional model and molecular dynamics simulation of the active site of the self-splicing intervening sequence of the bacteriophage T4 nrdB messenger RNA

Biochemistry ◽  
1990 ◽  
Vol 29 (45) ◽  
pp. 10317-10322 ◽  
Author(s):  
Lennart Nilsson ◽  
Agneta Aahgren-Staalhandske ◽  
Ann Sofie Sjoegren ◽  
Solveig Hahne ◽  
Britt Marie Sjoeberg
Keyword(s):  
Molecular Dynamics ◽  
Active Site ◽  
Messenger Rna ◽  
Bacteriophage T4 ◽  
Three Dimensional ◽  
The Self ◽  
Dynamics Simulation ◽  
Dimensional Model ◽  
Three Dimensional Model ◽  
Self Splicing

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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