Reverse transcriptases: Intron-encoded proteins found in thermophilic bacteria

Ng, Bernie; Nayak, Sunil; Gibbs, Moreland D.; Lee, Jun; Bergquist, Peter L.

doi:10.1016/j.gene.2007.02.003

Cited by 8 publications

(23 citation statements)

References 17 publications

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“…Interestingly, in four strains of thermophilic Geobacillus kaustophilus , G. stearothermophilus and Bacillus caldolyticus the essential housekeeping recA gene is interrupted by a group II intron (84,85). It has been suggested that the element serves as an intron in this locus rather than a selfish mobile retroelement (although it encodes an RT ORF) and that its activity might be related to some regulation of the recA gene expression under high temperature.…”

Section: Discussionmentioning

confidence: 99%

Survey of group I and group II introns in 29 sequenced genomes of the Bacillus cereus group: insights into their spread and evolution

Tourasse

Kolstø

2008

Nucleic Acids Research

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Group I and group II introns are different catalytic self-splicing and mobile RNA elements that contribute to genome dynamics. In this study, we have analyzed their distribution and evolution in 29 sequenced genomes from the Bacillus cereus group of bacteria. Introns were of different structural classes and evolutionary origins, and a large number of nearly identical elements are shared between multiple strains of different sources, suggesting recent lateral transfers and/or that introns are under a strong selection pressure. Altogether, 73 group I introns were identified, inserted in essential genes from the chromosome or newly described prophages, including the first elements found within phages in bacterial plasmids. Notably, bacteriophages are an important source for spreading group I introns between strains. Furthermore, 77 group II introns were found within a diverse set of chromosomal and plasmidic genes. Unusual findings include elements located within conserved DNA metabolism and repair genes and one intron inserted within a novel retroelement. Group II introns are mainly disseminated via plasmids and can subsequently invade the host genome, in particular by coupling mobility with host cell replication. This study reveals a very high diversity and variability of mobile introns in B. cereus group strains.

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Section: Discussionmentioning

confidence: 99%

Survey of group I and group II introns in 29 sequenced genomes of the Bacillus cereus group: insights into their spread and evolution

Tourasse

Kolstø

2008

Nucleic Acids Research

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“…GsI-IIB is a subgroup IIB2 intron that is inserted in the G. stearothermophilus recA gene and is related to previously described RT-encoding group II introns in the recA genes of Geobacillus kaustophilus (Chee and Takami 2005) and Bacillus caldolyticus (Ng et al 2007). GsI-IIC is a group IIC intron found in multiple copies in the G. stearothermophilus genome (Moretz and Lampson 2010).…”

Section: Recombinant Plasmidsmentioning

confidence: 97%

“…Group II intron RTs from thermophiles are expected to combine these useful properties with thermostability. Thus far, however, only one mobile group II intron RT, the LtrA protein encoded by the Ll.LtrB intron, has been expressed in bacteria and purified with high yield and activity (Saldanha et al 1999), while other group II intron RTs, including those from thermophiles, are poorly expressed and largely insoluble in the absence of bound RNAs (Vellore et al 2004;Chee and Takami 2005;Ng et al 2007). A further challenge for biotechnological development is that group II introns RTs often have mutations that decrease or abolish RT activity, reflecting that they are under selective pressure to suppress intron mobility, which is deleterious to their hosts (Mohr et al 2010).…”

Section: Introductionmentioning

confidence: 99%

“…Such MalE-rigid fusions typically have a linker region consisting of three to five alanine residues together with changes near the end of the MalE tag to replace charged amino acid residues with alanines. In initial experiments, we tested MalE rigid fusions of several group II intron RTs, including the mesophilic L. lactis Ll.LtrB intron RT (LtrA protein), several T. elongatus group II intron RTs that promote retrohoming at elevated temperatures in vivo (Mohr et al 2010), and two Geobacillus stearothermophilus group II intron RTs, which were previously difficult to express and purify with high yield and activity (Vellore et al 2004;Ng et al 2007). We found that group II intron RTs expressed as MalE rigid fusions (denoted MRF) support retrohoming in an E. coli plasmid assay (LtrA-MRF RT, 35% wild-type efficiency at 30°C; TeI4h * RT, 87% wild-type efficiency at 48°C ), indicating that they retain all required activities despite the presence of the MalE tag.…”

Section: Expression and Purification Of Group II Intron Rt Fusion Promentioning

confidence: 99%

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Thermostable group II intron reverse transcriptase fusion proteins and their use in cDNA synthesis and next-generation RNA sequencing

Mohr

Ghanem

Smith

et al. 2013

RNA

187

301

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Mobile group II introns encode reverse transcriptases (RTs) that function in intron mobility ("retrohoming") by a process that requires reverse transcription of a highly structured, 2-2.5-kb intron RNA with high processivity and fidelity. Although the latter properties are potentially useful for applications in cDNA synthesis and next-generation RNA sequencing (RNA-seq), group II intron RTs have been difficult to purify free of the intron RNA, and their utility as research tools has not been investigated systematically. Here, we developed general methods for the high-level expression and purification of group II intron-encoded RTs as fusion proteins with a rigidly linked, noncleavable solubility tag, and we applied them to group II intron RTs from bacterial thermophiles. We thus obtained thermostable group II intron RT fusion proteins that have higher processivity, fidelity, and thermostability than retroviral RTs, synthesize cDNAs at temperatures up to 81°C, and have significant advantages for qRT-PCR, capillary electrophoresis for RNA-structure mapping, and next-generation RNA sequencing. Further, we find that group II intron RTs differ from the retroviral enzymes in template switching with minimal base-pairing to the 3 ′ ends of new RNA templates, making it possible to efficiently and seamlessly link adaptors containing PCR-primer binding sites to cDNA ends without an RNA ligase step. This novel template-switching activity enables facile and less biased cloning of nonpolyadenylated RNAs, such as miRNAs or protein-bound RNA fragments. Our findings demonstrate novel biochemical activities and inherent advantages of group II intron RTs for research, biotechnological, and diagnostic methods, with potentially wide applications.

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“…The RTs also have a novel template-switching activity that enables facile attachment of adaptor sequences containing primer-binding sites and barcodes to cDNAs. These properties, combined with the availability of naturally occurring thermostable group II intron RTs [11,12] open new approaches for RNA-seq and the profiling and discovery of miRNAs and other non-coding RNAs [10,13]. …”

Section: Introductionmentioning

confidence: 99%

Biotechnological applications of mobile group II introns and their reverse transcriptases: gene targeting, RNA-seq, and non-coding RNA analysis

et al. 2014

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Mobile group II introns are bacterial retrotransposons that combine the activities of an autocatalytic intron RNA (a ribozyme) and an intron-encoded reverse transcriptase to insert site-specifically into DNA. They recognize DNA target sites largely by base pairing of sequences within the intron RNA and achieve high DNA target specificity by using the ribozyme active site to couple correct base pairing to RNA-catalyzed intron integration. Algorithms have been developed to program the DNA target site specificity of several mobile group II introns, allowing them to be made into ‘targetrons.’ Targetrons function for gene targeting in a wide variety of bacteria and typically integrate at efficiencies high enough to be screened easily by colony PCR, without the need for selectable markers. Targetrons have found wide application in microbiological research, enabling gene targeting and genetic engineering of bacteria that had been intractable to other methods. Recently, a thermostable targetron has been developed for use in bacterial thermophiles, and new methods have been developed for using targetrons to position recombinase recognition sites, enabling large-scale genome-editing operations, such as deletions, inversions, insertions, and ‘cut-and-pastes’ (that is, translocation of large DNA segments), in a wide range of bacteria at high efficiency. Using targetrons in eukaryotes presents challenges due to the difficulties of nuclear localization and sub-optimal magnesium concentrations, although supplementation with magnesium can increase integration efficiency, and directed evolution is being employed to overcome these barriers. Finally, spurred by new methods for expressing group II intron reverse transcriptases that yield large amounts of highly active protein, thermostable group II intron reverse transcriptases from bacterial thermophiles are being used as research tools for a variety of applications, including qRT-PCR and next-generation RNA sequencing (RNA-seq). The high processivity and fidelity of group II intron reverse transcriptases along with their novel template-switching activity, which can directly link RNA-seq adaptor sequences to cDNAs during reverse transcription, open new approaches for RNA-seq and the identification and profiling of non-coding RNAs, with potentially wide applications in research and biotechnology.

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Reverse transcriptases: Intron-encoded proteins found in thermophilic bacteria

Cited by 8 publications

References 17 publications

Survey of group I and group II introns in 29 sequenced genomes of the Bacillus cereus group: insights into their spread and evolution

Survey of group I and group II introns in 29 sequenced genomes of the Bacillus cereus group: insights into their spread and evolution

Thermostable group II intron reverse transcriptase fusion proteins and their use in cDNA synthesis and next-generation RNA sequencing

Biotechnological applications of mobile group II introns and their reverse transcriptases: gene targeting, RNA-seq, and non-coding RNA analysis

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