Preliminary structure analysis of the DH/PH domains of leukemia-associated RhoGEF

Kristelly, Romana; Earnest, Brett T.; Krishnamoorthy, Lakshmipriya; Tesmer, John J.G.

doi:10.1107/s0907444903018067

Cited by 20 publications

(19 citation statements)

References 13 publications

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“…pMalE-RT constructs (e.g., pMalE-TeI4c, pMalE-TeI4h * ) contain the indicated RT ORF with an N-terminal MalE tag cloned behind the tac promoter in pMal-c2t (derived from pMal-c2x; New England BioLabs) (Kristelly et al 2003). TeI4h…”

Section: Recombinant Plasmidsmentioning

confidence: 99%

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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Section: Recombinant Plasmidsmentioning

confidence: 99%

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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“…Tobacco etch virus protease (35,36) and CaM (37,38) were purified, and peptide substrate (Pep-S) (33) was synthesized as described earlier.…”

Section: Expression and Purification Of Proteinsmentioning

confidence: 99%

The Molecular Mechanism of Eukaryotic Elongation Factor 2 Kinase Activation

Tavares¹,

Ferguson

Giles

et al. 2014

Journal of Biological Chemistry

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Background: Eukaryotic elongation factor 2 kinase (eEF-2K) regulates protein translation elongation rates. Results: eEF-2K activation involves a two-step process of calmodulin binding and rapid Thr-348 autophosphorylation. Conclusion: Activation of eEF-2K involves two distinct allosteric steps, both of which potentially induce a conformational change. Significance: This mechanism provides a framework for understanding how eEF-2K integrates inputs from multiple upstream signaling pathways.

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“…The latter is a derivative of pMAL-c2x (New England Biolabs; Ipswich, MA) in which the factor Xa protease cleavage site between MalE and the expressed protein was replaced by a tobacco etch virus (TEV) protease-cleavage site. 45 To construct pMAL-Mss116p, the Mss116p ORF minus the mt targeting sequence (codons 1-36) was amplified by PCR of genomic DNA from yeast strain BY4704, 46 using an N-terminal primer, which amplified from codon 37 and added a 5′-BamHI site, and a C-terminal primer, which amplified from the TAG stop codon and added a 3′-HindIII site. The resulting PCR product was then cloned between the BamHI and HindIII sites of the vector.…”

Section: Recombinant Plasmidsmentioning

confidence: 99%

Involvement of DEAD-box Proteins in Group I and Group II Intron Splicing. Biochemical Characterization of Mss116p, ATP Hydrolysis-dependent and -independent Mechanisms, and General RNA Chaperone Activity

Halls

Mohr

Campo

et al. 2007

Journal of Molecular Biology

152

242

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The RNA-catalyzed splicing of group I and group II introns is facilitated by proteins that stabilize the active RNA structure or act as RNA chaperones to disrupt stable inactive structures that are kinetic traps in RNA folding. In Neurospora crassa and Saccharomyces cerevisiae, the latter function is fulfilled by specific DEAD-box proteins, denoted CYT-19 and Mss116p, respectively. Previous studies showed that purified CYT-19 stimulates the in vitro splicing of structurally diverse group I and group II introns and uses the energy of ATP binding or hydrolysis to resolve kinetic traps. Here, we purified Mss116p and show that it has RNA-dependent ATPase activity, unwinds RNA duplexes in a non-polar fashion, and promotes ATP-independent strand-annealing. Further, we show that Mss116p binds RNA non-specifically and promotes in vitro splicing of both group I and group II intron RNAs, as well as RNA cleavage by the aI5γ-derived D135 ribozyme. However, Mss116p also has ATP-hydrolysis-independent effects on some of these reactions, which are not shared by CYT-19 and may reflect differences in its RNA-binding properties. We also show that a non-mitochondrial DEAD-box protein, yeast Ded1p, can function almost as efficiently as CYT-19 and Mss116p in splicing the yeast aI5γ group II intron and less efficiently in splicing the bI1 group II intron. Together, our results show that Mss116p, like CYT-19, can act broadly as an RNA chaperone to stimulate the splicing of diverse group I and group II introns and that Ded1p also has an RNA chaperone activity that can be assayed by its effect on splicing mitochondrial introns. Nevertheless, these DEAD-box protein RNA chaperones are not completely interchangeable and appear to function in somewhat different ways using biochemical activities that have likely been tuned by coevolution to function optimally on specific RNA substrates.

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Preliminary structure analysis of the DH/PH domains of leukemia-associated RhoGEF

Cited by 20 publications

References 13 publications

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

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

The Molecular Mechanism of Eukaryotic Elongation Factor 2 Kinase Activation

Involvement of DEAD-box Proteins in Group I and Group II Intron Splicing. Biochemical Characterization of Mss116p, ATP Hydrolysis-dependent and -independent Mechanisms, and General RNA Chaperone Activity

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