scholarly journals A Functional Homing Endonuclease in the Bacillus anthracis nrdE Group I Intron

2007 ◽  
Vol 189 (14) ◽  
pp. 5293-5301 ◽  
Author(s):  
David Nord ◽  
Eduard Torrents ◽  
Britt-Marie Sjöberg
Keyword(s):  
Bacillus Anthracis ◽  
Homing Endonuclease ◽  
Insertion Site ◽  
Group I Intron ◽  
Coding Region ◽  
Recognition Sequence ◽  
Intron Insertion ◽  
Group I ◽  
Serovar Typhimurium ◽  
Self Splicing

ABSTRACT The essential Bacillus anthracis nrdE gene carries a self-splicing group I intron with a putative homing endonuclease belonging to the GIY-YIG family. Here, we show that the nrdE pre-mRNA is spliced and that the homing endonuclease cleaves an intronless nrdE gene 5 nucleotides (nt) upstream of the intron insertion site, producing 2-nt 3′ extensions. We also show that the sequence required for efficient cleavage spans at least 4 bp upstream and 31 bp downstream of the cleaved coding strand. The position of the recognition sequence in relation to the cleavage position is as expected for a GIY-YIG homing endonuclease. Interestingly, nrdE genes from several other Bacillaceae were also susceptible to cleavage, with those of Bacillus cereus, Staphylococcus epidermidis (nrdE1), B. anthracis, and Bacillus thuringiensis serovar konkukian being better substrates than those of Bacillus subtilis, Bacillus lichenformis, and S. epidermidis (nrdE2). On the other hand, nrdE genes from Lactococcus lactis, Escherichia coli, Salmonella enterica serovar Typhimurium, and Corynebacterium ammoniagenes were not cleaved. Intervening sequences (IVSs) residing in protein-coding genes are often found in enzymes involved in DNA metabolism, and the ribonucleotide reductase nrdE gene is a frequent target for self-splicing IVSs. A comparison of nrdE genes from seven gram-positive low-G+C bacteria, two bacteriophages, and Nocardia farcinica showed five different insertion sites for self-splicing IVSs within the coding region of the nrdE gene.

scholarly journals Group I Self-Splicing Intron in the recA Gene of Bacillus anthracis

2002 ◽  
Vol 184 (14) ◽  
pp. 3917-3922 ◽  
Author(s):  
Minsu Ko ◽  
Hyang Choi ◽  
Chankyu Park
Keyword(s):  
Bacillus Anthracis ◽  
Group I Intron ◽  
Reca Gene ◽  
Trna Genes ◽  
Group I Introns ◽  
Protein Coding ◽  
E Coli ◽  
Coding Regions ◽  
Group I ◽  
Self Splicing

ABSTRACT Self-splicing introns are rarely found in bacteria and bacteriophages. They are classified into group I and II according to their structural features and splicing mechanisms. While the group I introns are occasionally found in protein-coding regions of phage genomes and in several tRNA genes of cyanobacteria and proteobacteria, they had not been found in protein-coding regions of bacterial genomes. Here we report a group I intron in the recA gene of Bacillus anthracis which was initially found by DNA sequencing as an intervening sequence (IVS). By using reverse transcriptase PCR, the IVS was shown to be removable from the recA precursor mRNA for RecA that was being translated in E. coli. The splicing was visualized in vitro with labeled free GTP, indicating that it is a group I intron, which is also implied by its predicted secondary structure. The RecA protein of B. anthracis expressed in E. coli was functional in its ability to complement a recA defect. When recA-negative E. coli cells were irradiated with UV, the Bacillus RecA reduced the UV susceptibility of the recA mutant, regardless of the presence of intron.

Reverse splicing of a mobile twin-ribozyme group I intron into the natural small subunit rRNA insertion site

2005 ◽  
Vol 33 (3) ◽  
pp. 482-484 ◽  
Author(s):  
Å.B. Birgisdottir ◽  
S.D. Johansen
Keyword(s):  
Horizontal Transfer ◽  
Homing Endonuclease ◽  
Insertion Site ◽  
Group I Intron ◽  
Small Subunit ◽  
Group I Introns ◽  
Small Subunit Rrna ◽  
Homing Endonuclease Gene ◽  
Group I ◽  
Endonuclease Gene

A mobile group I intron containing two ribozyme domains and a homing endonuclease gene (twin-ribozyme intron organization) can integrate by reverse splicing into the small subunit rRNA of bacteria and yeast. The integration is sequence-specific and corresponds to the natural insertion site (homing site) of the intron. The reverse splicing is independent of the homing endonuclease gene, but is dependent on the group I splicing ribozyme domain. The observed distribution of group I introns in nature can be explained by horizontal transfer between natural homing sites by reverse splicing and subsequent spread in populations by endonuclease-dependent homing.

scholarly journals Role of Exonucleolytic Degradation in Group I Intron Homing in Phage T4

Genetics ◽  
1999 ◽  
Vol 153 (4) ◽  
pp. 1501-1512 ◽  
Author(s):  
Yi-Jiun Huang ◽  
Monica M Parker ◽  
Marlene Belfort
Keyword(s):  
Insertion Site ◽  
Rnase H ◽  
Nucleic Acid Metabolism ◽  
Exonuclease Activity ◽  
Intron Insertion ◽  
Vitro Assay ◽  
Phage T4 ◽  
Group I ◽  
Intron Homing

AbstractHoming of the phage T4 td intron is initiated by the intron-encoded endonuclease I-TevI, which cleaves the intronless allele 23 and 25 nucleotides upstream of the intron insertion site (IS). The distance between the I-TevI cleavage site (CS) and IS implicates endo- and/or exonuclease activities to resect the DNA segment between the IS and CS. Furthermore, 3′ tails must presumably be generated for strand invasion by 5′-3′ exonuclease activity. Three experimental approaches were used to probe for phage nucleases involved in homing: a comparative analysis of in vivo homing levels of nuclease-deficient phage, an in vitro assay of nuclease activity and specificity, and a coconversion analysis of flanking exon markers. It was thereby demonstrated that T4 RNase H, a 5′-3′ exonuclease, T4 DNA exonuclease A (DexA) and the exonuclease activity of T4 DNA polymerase (43Exo), 3′-5′ exonucleases, play a role in intron homing. The absence of these functions impacts not only homing efficiency but also the extent of degradation and flanking marker coconversion. These results underscore the critical importance of the 3′ tail in intron homing, and they provide the first direct evidence of a role for 3′ single-stranded DNA ends as intermediates in T4 recombination. Also, the involvement of RNase H, DexA, and 43Exo in homing provides a clear example of the harnessing of functions variously involved in phage nucleic acid metabolism for intron propagation.

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.

scholarly journals I-PpoI, the Endonuclease Encoded by the Group I Intron PpLSU3, Is Expressed from an RNA Polymerase I Transcript

1998 ◽  
Vol 18 (10) ◽  
pp. 5809-5817 ◽  
Author(s):  
Jue Lin ◽  
Volker M. Vogt
Keyword(s):  
Rna Polymerase ◽  
Homing Endonuclease ◽  
Group I Intron ◽  
Subcellular Fractionation ◽  
Rna Polymerase I ◽  
Rrna Genes ◽  
Temperature Sensitive ◽  
Group I ◽  
Polymerase I ◽  
Mutant Forms

ABSTRACT PpLSU3, a mobile group I intron in the rRNA genes of Physarum polycephalum, also can home into yeast chromosomal ribosomal DNA (rDNA) (D. E. Muscarella and V. M. Vogt, Mol. Cell. Biol. 13:1023–1033, 1993). By integrating PpLSU3 into the rDNA copies of a yeast strain temperature sensitive for RNA polymerase I, we have shown that the I-PpoI homing endonuclease encoded by PpLSU3 is expressed from an RNA polymerase I transcript. We have also developed a method to integrate mutant forms of PpLSU3 as well as theTetrahymena intron TtLSU1 into rDNA, by expressing I-PpoI in trans. Analysis of I-PpoI expression levels in these mutants, along with subcellular fractionation of intron RNA, strongly suggests that the full-length excised intron RNA, but not RNAs that are further cleaved, serves as or gives rise to the mRNA.

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
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