Protein splicing of the three Pyrococcus abyssi ribonucleotide reductase inteins

Kerrigan, Adam; Powers, Taryn L.; Dorval, Deirdre M.; Reitter, Julie N.; Mills, Kenneth V.

doi:10.1016/j.bbrc.2009.06.145

Cited by 7 publications

(11 citation statements)

References 23 publications

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“…This result should be viewed in the context of previous studies of inteins with noncanonical penultimate residues in archaea and chloroplasts that have shown disparate responses when mutated. Some of these unusual inteins have increased splicing when His replaces the native penultimate residue ( 51 , 52 ), some become splicing impaired ( 51 ), and others have no detectible change ( 53 ).…”

Section: Discussionmentioning

confidence: 99%

Mycobacteriophages as Incubators for Intein Dissemination and Evolution

Kelley

Lennon

Sea-Phages

et al. 2016

mBio

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Inteins are self-splicing protein elements that are mobile at the DNA level and are sporadically distributed across microbial genomes. Inteins appear to be horizontally transferred, and it has been speculated that phages may play a role in intein distribution. Our attention turns to mycobacteriophages, which infect mycobacteria, where both phage and host harbor inteins. Using bioinformatics, mycobacteriophage genomes were mined for inteins. This study reveals that these mobile elements are present across multiple mycobacteriophage clusters and are pervasive in certain genes, like the large terminase subunit TerL and a RecB-like nuclease, with the majority of intein-containing genes being phage specific. Strikingly, despite this phage specificity, inteins localize to functional motifs shared with bacteria, such that intein-containing genes have similar roles, like hydrolase activity and nucleic acid binding, indicating a global commonality among intein-hosting proteins. Additionally, there are multiple insertion points within active centers, implying independent invasion events, with regulatory implications. Several phage inteins were shown to be splicing competent and to encode functional homing endonucleases, important for mobility. Further, bioinformatic analysis supports the potential for phages as facilitators of intein movement among mycobacteria and related genera. Analysis of catalytic intein residues finds the highly conserved penultimate histidine inconsistently maintained among mycobacteriophages. Biochemical characterization of a noncanonical phage intein shows that this residue influences precursor accumulation, suggesting that splicing has been tuned in phages to modulate generation of important proteins. Together, this work expands our understanding of phage-based intein dissemination and evolution and implies that phages provide a context for evolution of splicing-based regulation.

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Section: Discussionmentioning

confidence: 99%

Mycobacteriophages as Incubators for Intein Dissemination and Evolution

Kelley

Lennon

Sea-Phages

et al. 2016

mBio

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“…1). Genomic DNA from Trichodesmium erythraeum IMS101 was a kind gift from Sonya Dyhrman, To improve expression of HMIGSsp, we made a mutation of Asp241Ala to reduce endonuclease activity [10]. Genomic DNA from Trichodesmium erythraeum IMS101 was a kind gift from Sonya Dyhrman, To improve expression of HMIGSsp, we made a mutation of Asp241Ala to reduce endonuclease activity [10].…”

Section: Methodsmentioning

confidence: 99%

“…HMIGSsp has HM, followed by the 15 C-terminal residues of the N-extein, the 332 residue Ssp Ter intein, the 13 N-terminal residues of the C-extein, and G. HMIGTer has HM, followed by the 17 C-terminal residues of the N-extein, the 336 residue Ter Ndse1 intein, the 13 N-terminal residues of the C-extein, and G. We generated the expression vectors by subcloning into pHMIGLon [9], using appropriate oligonucleotide primers to amplify the intein-containing genes by PCR from genomic DNA. Particularly in cases in which the unspliced precursor is the major protein product of HIMGSsp or HMIGTery, residual homing endonuclease activity likely decreases the yield of overexpressed protein [10]. Particularly in cases in which the unspliced precursor is the major protein product of HIMGSsp or HMIGTery, residual homing endonuclease activity likely decreases the yield of overexpressed protein [10].…”

Section: Methodsmentioning

confidence: 99%

“…[Mutation of the His residue to Arg does not affect splicing activity (data not shown)]. To improve expression of HMIGSsp, we made a mutation of Asp241Ala to reduce endonuclease activity [10]. Particularly in cases in which the unspliced precursor is the major protein product of HIMGSsp or HMIGTery, residual homing endonuclease activity likely decreases the yield of overexpressed protein [10].…”

Section: Methodsmentioning

confidence: 99%

“…To improve expression of HMIGSsp, we made a mutation of Asp241Ala to reduce endonuclease activity [10]. Particularly in cases in which the unspliced precursor is the major protein product of HIMGSsp or HMIGTery, residual homing endonuclease activity likely decreases the yield of overexpressed protein [10]. To study isolated C-terminal cleavage of the inteins, we made Cys1Ala mutations of both HMIGSsp and HMIGTer by site directed mutagenesis using appropriate primers.…”

Section: Methodsmentioning

confidence: 99%

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Coordination of the third step of protein splicing in two cyanobacterial inteins

et al. 2017

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The third step of protein splicing is cyclization of Asn coupled to peptide bond cleavage. In two related cyanobacterial inteins, this step is facilitated by Asn or Gln. For a Synechococcus sp. PCC7002 intein, the isolated third step of protein splicing is more efficient with its native Asn than with substitution to Gln. For a Trichodesmium erythraeum intein, its native Gln facilitates the third step as efficiently as with Asn. Despite these differences, the yield of splicing is not affected, suggesting that the third step is influenced by mechanism-linked conformational changes. A conserved catalytic His and the penultimate residue also play roles in promoting side-chain cyclization.

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Methods to Study the Structure and Catalytic Activity of cis-Splicing Inteins

Zhao

Wang

et al. 2020

Expressed Protein Ligation

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The autocatalytic process of protein splicing is facilitated by an intein, which interrupts flanking polypeptides called exteins. The mechanism of protein splicing has been studied by overexpression in E. coli of intein fusion proteins with nonnative exteins. Inteins can be used to generate reactive α-thioesters, as well as proteins with N-terminal Cys residues, to facilitate expressed protein ligation. As such, a more detailed understanding of the function of inteins can have significant impact for biotechnology applications. Here, we provide biochemical methods to study splicing activity and NMR methods to study intein structure and the catalytic mechanism.

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Protein splicing of the three Pyrococcus abyssi ribonucleotide reductase inteins

Cited by 7 publications

References 23 publications

Mycobacteriophages as Incubators for Intein Dissemination and Evolution

Mycobacteriophages as Incubators for Intein Dissemination and Evolution

Coordination of the third step of protein splicing in two cyanobacterial inteins

Methods to Study the Structure and Catalytic Activity of cis-Splicing Inteins

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